Neovascularization Induced by the Hyaluronic Acid-Based Spongy

Subscriber access provided by TRAKYA UNIVERSITESI

Article

Neovascularization induced by the hyaluronic acidbased spongy-like hydrogels degradation products Lucília Pereira da Silva, Rogerio P. Pirraco, Tircia C. Santos, Ramon Novoa-Carballal, Mariana T. Cerqueira, Rui L. Reis, Vitor M. Correlo, and Alexandra P. Marques ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b11684 • Publication Date (Web): 18 Nov 2016 Downloaded from http://pubs.acs.org on November 20, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Neovascularization Induced by the Hyaluronic Acid-Based Spongy-Like Hydrogels Degradation Products Lucília P. da Silva1,2, Rogério P. Pirraco1,2, Tírcia C. Santos1,2, Ramon NovoaCarballal1,2, Mariana T. Cerqueira1,2, Rui L. Reis1,2, Vitor M. Correlo1,2, Alexandra P. Marques1,2*. 1- 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark - Parque da Ciência e Tecnologia, 4805-017 Barco, Taipas, Guimarães, Portugal; 2- ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal, *[email protected], 00351 253 510 909

Abstract Neovascularization has been a major challenge in many tissue regeneration strategies. Hyaluronic acid (HA) of 3 to 25 disaccharides is known to be angiogenic due to its interaction with endothelial cell receptors. This effect has been explored with HA-based structures but a transitory response is observed due to HA burst biodegradation. Herein we developed gellan gum (GG)-HA spongy-like hydrogels from semi-interpenetrating network hydrogels with different HA amounts. Enzymatic degradation was more evident in the GG-HA with high HA amount due to their lower mechanical stability, also resulting from the degradation itself, which facilitated the access of the enzyme to the HA in the bulk. GG-HA spongy-like hydrogels hyaluronidase-mediated degradation lead to the release of HA oligosaccharides of different amounts and sizes in a HA content-dependent manner which promoted in vitro proliferation of human umbilical cord vein endothelial cells (HUVECs) but not their migration. Although no effect was observed in human dermal microvascular endothelial cells (hDMECs) in vitro, the implantation of GG-HA spongy-like hydrogels in an ischemic hindlimb mice model promoted neovascularization in a material-dependent manner, consistent with the in vitro degradation profile. Overall, GG-HA spongy-like hydrogels with a sustained release of HA oligomers are valuable options to improve tissue vascularization, a critical issue in several applications in the tissue engineering and regenerative medicine field. 1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 32

Keywords: neovascularization, angiogenesis, hyaluronic acid, gellan gum spongy-like hydrogels, ischemia, biodegradation

1. Introduction The success of many tissue regeneration strategies greatly relies on the ability of a biomolecule, a biomaterial or a tissue engineered construct to promote neovascularization in a relatively short period of time post implantation1–4. Neovascularization has been pursued by the in situ delivery of growth factors4–8 and/or endothelial cells and undifferentiated adult stem cells9–12 taking advantage of their angiogenic potential, as well as by the transplantation of pre-vascularized constructs benefiting inosculation12,13. Nonetheless, all of these strategies have issues that are still to be addressed. Insufficient retention of angiogenic factors in the area of interest and at an effective concentration are some of the limitations of growth factors-based approaches. Cellular strategies are restricted by the endothelial cell’s availability and reduced in vitro proliferative capacity, and by the still unrevealed mechanisms of action of stem cells. High molecular weight hyaluronic acid (HA) is a ubiquitous, abundant and structural constituent of the extracellular matrix. During development and in tissues under remodeling or healing, low molecular weight HA fragments are originated by hyaluronidase-mediated biodegradation. These fragments are known to have a crucial role in activating different cell signaling pathways14,15. The interaction of HA oligomers (3 up to 25 disaccharides) with endothelial cells, mediated by the HA binding to CD4416–18 and to receptor for HA-mediated cell motility (RHAMM)19, was shown to promote in vitro endothelial cell proliferation, migration, collagen synthesis and cell sprouting20–24, as well as in vivo22–26 and ex vivo neovascularization21,27. A wide range of HA-based biomaterials28 and in combination with angiogenic growth factors29–33 or adult cells32,34,35, have shown to promote angiogenesis and consequently neovascularization. Nonetheless, a major limitation of all these strategies relates to the fast degradation of the HA which leads to a transient neovascularization36–41. This is supported by a work that 2 ACS Paragon Plus Environment

Page 3 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


showed prolonged neovascularization due to the slow release of 6.5 KDa HA oligomers encapsulated within a porous collagen scaffold42. Gellan gum (GG) is, an anionic polysaccharide produced by Sphingomonas elodea that forms hydrogels by temperature decrease and ionic crosslinking. We have previously shown that by processing GG-based hydrogels through a highly controlled and sequential methodology we generate spongy-like hydrogels, which have properties between hydrogels and sponges43,44. Spongy-like hydrogels have different and tunable physical, morphological and mechanical properties relatively to the precursor hydrogels. Moreover, are formed after hydration of dried polymeric networks with a solvent/solution, with/without cells or bioactive molecules, which provides great versatility. Unlike precursor hydrogels, spongy-like hydrogels support the adhesion of entrapped/encapsulated cells in the absence of functionalization of the material/structure with cell adhesive features. This intrinsic cell-adhesive character benefitted the action of human dermal and epidermal cells, as well as endothelial cells in the re-epithelialization and neovascularization of fullthickness full-thickness skin wounds45,46. Based on these results in this work we hypothesized that GG-HA spongy-like hydrogels composition affected the degradation profile leading to a differentiated release of hyaluronic acid oligomers and consequently to different levels of neovascularization. Thus, GG-HA spongylike hydrogels with 0.25 % or 0.75 % HA were produced and characterized in terms of microstructure, mechanical performance under compression and water content. These features were then correlated with the GG-HA spongy-like hydrogels biodegradation profiles as well as with the type and amount of released HA fragments. The in vitro effect of the degradation products of GG-HA spongy-like hydrogels over micro and macrovascular endothelial cells proliferation and migration was studied prior assessing the potential of GG-HA spongy-like hydrogels over neovascularization in an ischemic hind limb mice model.

3 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 32

2. Experimental 2.1. Spongy-like hydrogels preparation GG-HA hydrogels were prepared as previously described with some modifications44. Briefly, HA (1.5 MDa, LifeCore, USA) was dissolved in deionized water under stirring at room temperature for 3 h. Gelzan powder (SIGMA, Portugal) was then added to the HA solution, which was kept under stirring at 90ºC for 30 min. Two formulations of GG-HA were prepared: GG-HA 1 % (0.25 % HA / 0.75 % GG) and GG-HA 2 % (0.75 % HA / 1.25 % GG). The polymeric solution was then casted into molds and rapidly mixed with α-minimum essential medium (MEM Life Technologies, Scotland) for crosslinking. The hydrogel was progressively formed until room temperature was reached. Spongy-like hydrogels were prepared from these hydrogels after stabilization in phosphate buffered saline (PBS) solution for 28 h, freezing at -80°C, freeze-drying (LyoAlfa 10/15, Telstar, Spain) for three days to obtain GG-HA dried polymeric networks, and re-hydration. 2.2. Scanning Electron Microscopy A Leica Cambridge S360 microscope (UK) was used to analyze the microarchitecture of the dried polymeric networks, after coating with gold (Cressington Sputter Coater), at an accelerating voltage of 15 kV. A JEOL JSM 6301F/Oxford

INCA

Energy

350/Gatan

Alto

2500

microscope

(CEMUP

laboratories, Porto, Portugal) was used to analyze the microarchitecture of the hydrogels and spongy-like hydrogels. 2.3. Micro-computed tomography Dried polymeric networks microarchitecture was analyzed using a highresolution X-Ray Microtomography (micro-CT) System Skyscan 1072 scanner (Skyscan, Kontich, Belgium). Representative data sets of 150 slices were transformed into a binary picture using a dynamic threshold of 45e255 (grey values) to distinguish polymeric material from pore voids. This data was used for morphometric analysis (CT Analyser v1.5.1.5, SkyScan), which included quantification of the pore wall thickness, structure porosity and pore size. 3D virtual 4 ACS Paragon Plus Environment

Page 5 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


models of representative regions in the bulk of the materials were created, visualized and registered using the image processing software (CT-vox, SkyScan). 2.4. Compression tests The mechanical behavior of spongy-like hydrogels was tested under static compression. Dried polymeric networks were immersed into PBS for 24 h at room temperature for complete re-hydration, before testing. The unconfined static compressive mechanical properties of the samples were measured using an INSTRON 5543 (Instron Int. Ltd., USA). Samples (10x6 mm) were submitted to a pre-load of 0.1 N before testing and were tested up to 60 % of strain, at a loading rate of 2 mm/min. 2.5. Water content analysis Dried polymeric networks were weighted (W d) and then immersed into PBS for 48 h at 37ºC. After 48 h the superficial water was removed with a filter paper and the obtained spongy-like hydrogels were weighted (W w). Water content was obtained using the following Equation 1. Equation 1 – Water content (%) = (W w – W d)/Wd x 100 2.6. Degradation tests The degradation of spongy-like hydrogels was analyzed after immersion of the dried polymeric networks for 1, 7, 14, 21 and 28 days at 37ºC and 180 rpm into a PBS solution containing 0.2 g/L NaN3 (SIGMA, USA), with or without 50/150 U/mL hyaluronidase type IV (SIGMA, Portugal). Solutions were replenished every 3/4 days and the supernatants containing the cumulative degradation products were collected, frozen and freeze-dried. Prior analysis the HA fragments (HA-F) were either re-suspended in the GPC eluent for the GPC analysis or in EGM medium for the in vitro cell tests, and filtered through a 0.22 µm membrane. 2.7. Mass loss analysis Dried polymeric networks were weighted (W i) and then immersed into the degradation solution. The formed spongy-like hydrogels were weighted at each



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 32

time point (Wf) after freeze-drying. Mass loss was calculated according to the Equation 2, as follows. Equation 2 - Mass loss (%) = (Wi-Wf)/Wi x 100 2.8. Dinitrosalicylic Acid assay The amount of degraded polysaccharides in the solutions collected from the degradation assay was evaluated by quantifying the reducing sugars at the end of the HA chains through the Dinitrosalicylic Acid (DNS) assay47. Briefly, 50 µL of dinitrosalicylic acid reagent (SIGMA, Portugal) and 50 µL of sample were mixed and boiled for 5 min at 100ºC. Subsequently, 500 µL of deionized water were added to each mixture and then, 100 µL of the obtained solution was plated into each well of a 96 well-plate. The absorbance was read at 540 nm in a Synergy HT multi-mode microplate reader (Synergy HT, BioTek, USA) and the concentration of reducing sugars was calculated according to a dextran standard curve ranging from 0.1 to 2 mg/mL. 2.9. Gel Permeation/Size Exclusion Chromatography Gel Permeation Chromatography/Size Exclusion Chromatography (Viscotec TDA 305, Malvern, England) was used to determine the molecular weight of HA-F. The column set was composed by a pre-column Suprema 5 µm 8x50 S/N 3111265, Suprema 30 Å 5 µm 8x300 S/N 3112751, Suprema 1000 Å 5 µm 8x300 S/N 3112851 PL and Aquagel-OH MIXED 8 µm 7.5x300 S/N 8M-AOHMIX-46-51, with refractive index detection (RI-Detector 8110, Bischoff). The system was kept at 30ºC and 0.01 M NaH2PO4 in 0.1 M NaN3 at pH 6.7 was used as eluent of HA-F (1 mg/mL) at the rate of 1 mL/min. A calibration curve was built using glucose (MP 180) and pullulan standards of

known molecular weight (Pullulan MP 5.900,

Pullulan MP 11.100, Pullulan MP 21.100, Pullulan MP 47.100, Pullulan MP 107.000, Pullulan MP 200.000, Pullulan MP 375.000, Pullulan MP 708.000, PSS GmbH, Germany) using the refractive index detector. 2.10.

Cell culture

Human umbilical cord vein endothelial cells (HUVECs) were purchased from Lonza (USA) and human dermal microvascular endothelial cells (hDMECs) were 6 ACS Paragon Plus Environment

Page 7 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


isolated from human adult skin samples, obtained from healthy donors undergoing abdominoplasties at Hospital da Prelada (Porto), after patient’s informed consent and 3B’s Research Group/Hospital ethical committees approval, as previously described48. Cells were routinely cultured in flasks coated with gelatin type A (0.7 % w/v, SIGMA, Portugal) in EndoGRO-VEGF and EndoGRO-MV-VEGF Complete Media (Millipore, USA) for HUVECs and hDMECs, respectively. 2.11.

Immunocytochemistry

In order to characterize the expression of HA receptors, HUVECs and hDMECs (5.000 cells/cm2) were seeded on gelatin-coated coverslips and after overnight adhesion, fixed with 10 % of formalin. Cells were permeabilized with a cold solution of 1 % of Triton X (SIGMA, Portugal) for 20 min and non-specific antigen bounding was blocked with 2.5 % of horse serum (Vector Laboratories, USA) for 30 min. Subsequently, cells were incubated with the fluorescent labeled antibody anti-human CD44-PE (BD Biosciences, Germany) or goat anti-human CD168 (Santa Cruz, USA) overnight at 4ºC. For the detection of CD168, samples were further incubated with Alexa Fluor 488 secondary antibody. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, SIGMA, USA). Samples were observed using an AxioImager Z1m microscope (Zeiss, Germany) and images acquired with Zen 2012 software. 2.12.

DNA Quantification

To assess the effect of HA-F over cells proliferation, HUVECs (500 cells/cm2) and hDMECs (2500 cells/cm2) were seeded on gelatin-coated 24-well plates and left to adhere overnight. HA-F were diluted in EndoGRO-VEGF or EndoGRO-MV media and respectively added to HUVECs and hDMECs cultures, at a concentration of 0.2 and 2 mg/mL in a total volume of 500 µL. HA with a molecular weight of 1.5 MDa was used at the same concentrations as the HA-F, as a control condition. After 7 days, the media were removed, cells were washed with PBS and kept in 1 mL of ultra-pure water prior freezing at -80ºC for cell membrane disruption. DNA quantification was performed in the cell lysates using the



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 32

Picogreen dsDNA assay kit (Life Technologies, Scotland) following manufacturer’s instructions.

2.13.

In vitro migration assay

Cell migration was evaluated using the Boyden Chamber assay. Briefly, HUVECs and hDMECs (10.000 cells) were seeded on the gelatin-coated transwell cell culture inserts (VWR, Portugal). HA-F or 1.5 MDa HA were added to the lower part of the transwell (8 µm pore size, Corning Life Sciences, USA) at the respective concentrations. After 4 h, the media inside the inserts and the non-migrating cells were removed with cotton-tipped swabs. After carefully cleaning the inserts with PBS, cells on the lower part of the insert were fixed with 10 % of formalin for 20 min and stained with DAPI for 30 min, all at RT. Cells that migrated to the bottom of the insert were counted using a fluorescence inverted microscope (Axio Observer, Zeiss, Germany). 2.14.

Implantation in ischemia hind limb

The implantation procedure was approved by the Direcção Geral de Alimentação Veterinária (DGAV), the Portuguese National Authority for Animal Health, and all the surgical procedures respected the national regulations and international animal welfare rules, according to the Directive 2010/63/EU. Twelve animals were divided in three groups: with GG-HA 1 % spongy-like hydrogels (GGHA 1 % group), with GG-HA 2 % spongy-like hydrogels (GG-HA 2 % group) and no material (control). In detail, C57Bl/6 mice (Harlan, Spain) were anaesthetized with an i.p injection of a mixture of ketamine (75 mg/Kg, Imalegene, Merial, France) and metedomidine (1 mg/kg, Domitor, Orion Pharma, Finland). The entire hind limb was shaved and sterilized prior incision through the dermis. The external iliac and the femoral arteries and veins were linked using 3-0 Ethilon (Ethicon, Somerville, NJ) and then the vessels were severed between the connection points where the materials were implanted. During the experiments, mice were kept under standard conditions in a 12 h light/dark cycle, with water and a commercial mice diet ad


Page 9 of 32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


libitum. After 7 weeks, animals were sacrificed and materials with the surrounding tissue were collected for analysis.

2.15.

Immunohistochemistry

The collected tissues were fixed in 10 % of formalin (Thermo Scientific, USA), dehydrated, embedded in paraffin (Thermo Scientific, USA), and cut into 3.5 µm sections. Tissue sections were stained with Trichrome Masson’s kit (SIGMA, Portugal) following a routine protocol. For immunohistochemistry, sequential tissue sections were deparaffinized in xylene, re-hydrated and boiled for 5 minutes in sodium citrate buffer (10 mM Sodium Citrate, 0.05 % Tween 20 (Bio-Rad, Netherlands), pH 6) for antigen retrieval. Afterwards, sections were incubated with 3 % of hydrogen peroxide (VWR, Portugal) for 20 min to block endogenous peroxidase activity, and with 2.5 % of horse serum (VECTASTAIN Elite ABC Kit, Vector Labs, USA) to block non-specific antigen binding. Samples were then incubated with the rabbit anti-mouse CD31 (1:15 dilution, ab28364 Abcam, UK) or rabbit anti-mouse α-SMA (1:100 dilution, ab5694 Abcam, UK) primary antibodies for 2 h at room temperature. For the detection of the primary antibodies, R.T.U. VECTASTAIN Elite ABC (Vector Labs, USA) and Peroxidase Substrate (DAB, Vector Labs, USA), kits were used according to the manufacturer’s instructions. Nuclei were stained with hematoxylin (BioOptica, Italy). Tissue sections were observed as described for the immunocytochemistry samples. 2.16.

Microvessel quantification

Microvessel density was determined in tissue sections immunostained for CD31 and α-SMA. Positively stained blood vessels were counted manually by four independent examiners and only CD31 or α-SMA stained cells integrated in vessels were considered. A minimum of five fields from each tissue section, inbetween the connection points, comprising the muscle and adipose tissue (control) or the neighboring areas of the implanted material (experimental) were analyzed.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 32

Results are presented as an average of the counted fields and expressed as microvessels per square millimeter. 2.17.

Blood perfusion analysis

Hind limb blood flow was measured using a Laser Doppler Perfusion Imaging (LDPI) (Moor Instruments, Model LDI2-IR, UK). Blood perfusion measurements were achieved by scanning the region of interest (ROI) of both limbs of anesthetized and shaved animals, under the same conditions of temperature and light, before and after surgery, and every week post-surgery. Results are presented as the ratio of the results of the ischemic to the normal limb. Ratio of 1 before surgery indicates equal blood perfusion in both limbs. Representative hind limb images are presented in a blood perfusion chromatic scale in which low to no flow is displayed as dark blue and high blood flow is displayed as red to white. 2.18.

Statistical Analysis

GraphPad software was used to perform statistical analysis. Data was analyzed by Shapiro-Wilk normality test. Data with normal distribution was analyzed using Two-way ANOVA with Bonferroni post-test; data that did not follow a normal distribution was analyzed by the Kruskal-Wallis test with Dunn’s Multiple Comparison post-test or two-tailed Mann Whitney test. Significance was set to *p

Neovascularization Induced by the Hyaluronic Acid-Based Spongy

Recommend Documents