Bioactive Injectable Hydrogels Containing Desferrioxamine and

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Biological and Medical Applications of Materials and Interfaces

Bioactive Injectable Hydrogels Containing Desferrioxamine and Bioglass for Diabetic Wound Healing Lingzhi Kong, Zhi Wu, Huakun Zhao, Haomin Cui, Ji Shen, Jiang Chang, Haiyan Li, and Yaohua He ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b09191 • Publication Date (Web): 16 Aug 2018 Downloaded from http://pubs.acs.org on August 17, 2018

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ACS Applied Materials & Interfaces

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Bioactive Injectable Hydrogels Containing Desferrioxamine and

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Bioglass for Diabetic Wound Healing

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Lingzhi Kong1#, Zhi Wu2, 3#, Huakun Zhao1, Haomin Cui1, Ji Shen1, Jiang Chang3, 4, Haiyan Li2, 3*,

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Yaohua He1*

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600 Yishan Road, Shanghai 200233, China.

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2

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Engineering, Shanghai Jiao Tong University, 1954 Huashan Road, Shanghai 200030, China.

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Department of Orthopedics, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital,

Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, School of Biomedical

School of Biomedical Engineering, Shanghai Jiao Tong University, 1954 Huashan Road,

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Shanghai 200030, China.

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4

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of Ceramics, Chinese Academy of Sciences, 1295 Dingxi Road, Shanghai 200050, China.

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State Key Laboratory of Performance Ceramics and Superfine Microstructure, Shanghai Institute

The two authors contributed to the work equally.

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Corresponding authors

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Yaohua He, Professor, MD

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Department of Orthopedics, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital

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E-mail: [email protected]

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Tel: +86 21 24058037

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Haiyan Li, Professor, PhD

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School of Biomedical Engineering, Shanghai Jiao Tong University

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Email: [email protected]

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Tel: +8618717902901

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ACS Paragon Plus Environment

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Abstract

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Diabetic wound is hard to heal mainly because of the difficulty in vascularization in the

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wound area. Accumulating evidences have shown that desferrioxamine (DFO) can promote

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secretion of hypoxia inducible factor-1 (HIF-1α), thereby upregulating the expression of

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angiogenic growth factors and facilitating revascularization. Our preliminary study has

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demonstrated that Si ions in bioglass (BG) can upregulate vascular endothelial growth factor

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(VEGF) expression, thus promoting revascularization. It is hypothesized that the combined use of

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BG and DFO may have a synergistic effect in promoting VEGF expression and revascularization.

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To prove this, we first determined DFO concentration range that had no apparent cytotoxicity on

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human umbilical vein endothelial cells (HUVECs). Then, the optimal concentration of DFO

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promoting tube formation of HUVECs was determined by cell migration and tube formation

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assays. In addition, we demonstrated that combination use of BG and DFO improved the

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migration and tube formation of HUVECs as compared with the use of either BG or DFO alone as

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BG and DFO could synergistically upregulated VEGF expression. Furthermore, a sodium alginate

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hydrogel containing both BG and DFO was developed and this hydrogel better facilitated diabetic

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skin wound healing than the use of either alone as BG and DFO in the hydrogels worked

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synergistically in promoting HIF-1α and VEGF expression and subsequently vascularization in the

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wound sites. Therefore, in this study, the synergistic effect in promoting revascularization between

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BG and DFO was first demonstrated and an injectable hydrogel simultaneously containing BG

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and DFO was developed for enhancing repair of diabetic chronic skin defects by taking

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advantages of the synergistic effects of BG and DFO in promoting revascularization. The study

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opens up a new prospect for the development of skin repair-promoting biomaterials.

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Keywords: Bioglass, Desferrioxamine, Injectable hydrogel, Vascularization, Diabetic wound

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1. Introduction

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World has witnessed a constant rise in the incidence of diabetes in recent years due to dietary

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and lifestyle changes, population aging, accelerated economic development and urbanization.

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Hyperglycemia in diabetes may lead to microvascular endothelial injury, vascular diastolic

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dysfunction and impaired ability of cells to carry oxygen to tissues. This may result in tissue

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ischemia, hypoxia and delay wound healing1-3. That is why wound healing is difficult in diabetic

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patients and chronic wound surface may be formed. Cell migration and proliferation, deposition


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and shaping of extracellular matrix are important processes in wound healing4-5. But all of them

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depend on the supply of oxygen, nutrients and cell factors to the cells by blood vessels. Therefore,

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promoting revascularization in the wound area is crucial for facilitating the healing of chronic

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wound surface. Up to now, vascular endothelial growth factor (VEGF) and basic fibroblast growth

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factor (bFGF) have been extensively applied alone or combined with scaffolds for angiogenesis in

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tissue engineering6-8. However, the use of growth factors in tissue engineering has also been

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limited by several shortcomings, such as expensive price for factors, the rigorous conditions for

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factors delivery or preservation, too short half-life period and uncontrollable release in vivo. By

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contrast, inorganic materials with bioactivity (such as silicate ceramics) and drugs can be applied

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in tissue engineering by overcoming all those obstacles.

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Desferrioxamine (DFO) is a natural product extracted from the fermentation liquor of

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Streptococcus spp. DFO is dissolvable in water and stable in aqueous solution. It has been

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reported that DFO promotes the secretion of VEGF and stromal cell-derived factor 1 by inducing

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the formation of hypoxia inducible factor-1α (HIF-1α), thereby facilitating vascularization9-11. A

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recent study has confirmed that DFO and drug carriers for sustained release of DFO can accelerate

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revascularization and wound healing12-14. Bioglass (BG) is a bioactive ceramic material containing

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Ca and Si ions. BG possesses excellent biological activity15, and ionic products formed by its

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degradation can promote osteogenesis16-17, angiogenesis and wound healing18-20. Among the ionic

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products of BG, Si ions can stimulate the gap junction communication between HUVECs and

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upregulated connexin43 (Cx43) expression, which results in stimulating of vascularization21.

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Moreover, the expression of vascular endothelial cadherin, bFGF, VEGF as well as their receptors

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in HUVECs, all of which are related to vascularization, can be promoted by BG ionic products21-22.

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Since revascularization is important for the repair of chronic skin wound, BG is considered as a

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promising bioactive material for promoting angiogenesis.

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In a recent study, Lin et al. used Si and Sr in combination and confirmed their synergistic

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effect in promoting osteogenesis23. Our preliminary study has also proved a synergic stimulatory

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effect of the combination of Cu and Si ions during vascularization24. However, all of these studies

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are combination use of bioactive materials or ions. Since both DFO and BG possess angiogenic

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effects, we proposed the combined use of DFO (drug) and BG (bioactive materials) to promote

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revascularization. However, DFO and BG usually exist in the form of powder, and their mixture



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may not be readily applied to the wound. In addition, BG powder will rapidly release Ca and Si

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ions in liquid, leading to a high pH value and causing pain to the patients25-27. DFO powder, when

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directly used, will also cause cytotoxicity due to an excessively high local concentration. Besides,

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wound healing requires a moist environment, and a large number of studies have shown that

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hydrogel is a good wound dressing28-30. Therefore, development a system that can provide a moist

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environment and functional DFO and BG ionic products is crucial for enhancing wound healing.

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Here we mixed DFO and BG in a hydrogel, which not only creates a moist environment for

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wound healing, but also serves as a carrier for the sustained release of DFO and BG. This method

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can properly control the concentration of DFO and ionic products of BG. The hydrogel consists of

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sodium alginate (SA), BG, DFO and gluconolactone (GDL). SA hydrogel has been widely used as

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wound dressing31-32, though it is usually not injectable and does not possess the functions of

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promoting angiogenesis and wound healing. In our previous experiment, we have developed an

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injectable hydrogel that contains the SA and bioactive silicate ceramic by using GDL33 sincea

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weak acidic environment can be created by GDL34-36 in the hydrogel, which allows for a sustained

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release of Ca ions from the bioactive silicate ceramic and in-situ crosslink SA to form an

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injectable hydrogel. In addition, the Si ions from the bioactive silicate ceramic can provide

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bioactivity for osteogenesis and angiogenesis33. Since BG is also a bioactive silicate ceramic, it

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can be reasoned that SA-BG-GDL system can also be injectable. Therefore, in this study, we

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further prepared an injectable and bioactive hydrogel that contains both BG and DFO by adding

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DFO into SA-BG-GDL injectable hydrogel system. This hydrogel was then applied to the animal

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model of wound healing to determine whether the combined use of BG and DFO in vivo has an

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enhanced promoting effect on revascularization and wound repair.

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2. Materials and Methods

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2.1. Cell isolation and culture

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Human umbilical venous endothelial cells (HUVECs) were isolated from human umbilical

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cord veins using the method reported by Bordenave et al.37.The isolated cells were dispersed,

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followed by culture with extracellular matrix in a humidified incubator which contained 5% CO2

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under 37oC. Endothelial culture medium (ECM) was used to culture HUVECs. Only the HUVECs

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of early passages (passage 2 ~ 7) were taken for the subsequent experiments.

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2.2. Effective DFO concentration determination


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Deferoxamine mesylate salt (DFO; > 99.0%) was purchased from Sigma-Aldrich. To assess

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the cytocompatibility of DFO with HUVECs, we performed CCK-8 assay. The cells were seeded

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to a 96-well plate at a density of 3×103 cells per well. Then, the cells were cultured in ECM

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containing different concentrations of DFO. At day 0, day 1, day 3 and day 5 of culture, 100µL of

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10% CCK-8 solution was added into each well and the cells were further cultured for 2hours

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before absorbance at 450nm of cells was measured with a UV spectrophotometer. By

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cytocompatibility assay, a cytocompatible DFO concentration range was obtained and used for the

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following two experiments.

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The transwell assay was employed to assess the function of DFO in the migration of

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HUVECs. In details, 5 ×104 cells were seeded in the upper chamber of a 24-well transwell plate

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(Corning; pore size = 8 µm) and cultured with ECM. Then, into the lower chamber, 600 µL of

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ECM with different concentrations of DFO was loaded. The cells were removed from the upper

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chamber with a cotton swab 8 hours later. Next, 4% paraformaldehyde was used to fix the cells

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migrating to the lower chamber, followed by 0.5% crystal violet staining for 10 min. Optical

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microscopy (provided by Leica, Germany) was used to observe the cells.

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For tube formation assay, into a 48-well plate, 100µL of Matrigel (Becton Dickinson, MA)

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was added, followed by gel under 37oC for 30 min. The suspension of cells (3×104 cells per well)

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was added onto Matrigel, and media containing different concentrations of DFO (4, 2, 1 or 0.5 µM)

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were used to treat the cells. Optical microscopy was employed to observe the tube formation

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following 6 hours incubation. The parameters and symbols of angiogenesis included nodes and

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tubes, which indicated the early and advanced stages, respectively. The number of nodes and tubes

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was manually counted based on the manufacturer’s instructions. After the proliferation, migration

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and tube formation experiments, an effective DFO concentration was determined.

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2.3. Effective BG ion extract preparation

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Bioactive Glass (BG) powders used in this research were kindly provided by Shanghai

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Institute of Ceramics, Chinese Academy of Science, which have a mean diameter of 20.28 µm (90%

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< 34.86 µm). In our preliminary studies, Si ion concentration of 0.7 ~ 1.8 µg/mL in BG ion

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extracts had the highest promoting effects on revascularization18,38. Therefore, BG ion extracts

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were prepared according to the procedures reported in literatures39-41. Then, the obtained ion

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extracts were diluted with cell culture medium to a final Si ion concentration of 1 µg/mL detected



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by inductively coupled plasma atomic emission spectroscopy (ICP-AES; Optima 3000DV,

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PerkinElmer, USA) for cell cultures.

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2.4. Effects of DFO, BG and BG+DFO on cell behaviors

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Once the proper DFO and BG ion extraction concentration were determined, CCK-8 assay

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was performed again to assess the cytocompatibility of BG ionic products, DFO and combination

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of BG and DFO (BG+DFO). Four different cell culture media was used in the following four

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groups: ECM (control group), ECM containing1 ug/mL Si ions of BG ionic products (BG group),

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ECM containing 2 µM DFO (DFO group), and ECM containing combination 1 ug/mL Si ions of

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BG ionic products and 2 µM DFO (BG+DFO group). The procedures were the same as above.

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To determine the influence BG ionic products, DFO and BG+DFO on migration and tube

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formation capacities of HUVECs, transwell assay and tube formation assay were performed again

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following the same procedures as above. HUVECs were cultured with the above four different

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culture media. The observation time was 4 hours in the tube formation assay.

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2.5. qRT-PCR

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To determine the influences of BG, DFO and BG+DFO on the gene expressions of HIF-1α,

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and VEGF in HUVECs, qRT-PCR was carried out. HUVECs were seeded to the 6-well plate at a

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density of 4×105 per well and cultured by the above four different cell culture media. RNA

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extraction was performed with Trizol reagent (Takara, Japan) from different cell samples 3 days

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later. Then, cDNA was synthesized by M-MLV (vazyme, China) and qRT-PCR were conducted by

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ChamQ SYBR qPCR Master Mix (vazyme, China). Normalization of the gene expression level to

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that of GAPDH was carried out, and the 2-△△Ct method was employed to analyze the relative gene

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expression. Each experiment was repeated for three times to calculate the average value. Primes

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used in this study were shown in supplementary file (Table S1).

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2.6. ELISA

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To determine the influences of BG, DFO and BG+DFO on the protein expressions of HIF-1α

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and VEGF, ELISA was carried out. HUVECs were seeded to the 6-well plate at a density of 4×105

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per well and cultured with the above four different cell culture media for 3 days. To determine the

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HIF-1α and VEGF protein concentrations in cells, HUVECs were collected after being cultured

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for 3 days and rinsed twice with sterile PBS containing protease inhibitors. A RIPA buffer (50 mM

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Tris·HCl, pH 8, 150 mM NaCl, 0.1% (vol/vol) Nonidet P-40, 10 µg/mL aprotinine, 10 µg/mL


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leupeptin, and 1mM AEBSF) was used to lyse the cells at 4°C for 30 min under agitation. Then,

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supernatant was obtained by centrifuging the lysates at 16,000 rpm and 4 °C for 20 min. The total

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HIF-1α and VEGF concentration in the cell lysates were then evaluated using a HIF-1α ELISA kit

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(Human/Mouse Total HIF-1α; Thermo Fisher Scientific, USA) and a Human VEGF Quantikine

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ELISA kit (Thermo Fisher Scientific, USA), respectively, according to manufacturer’s instructions.

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Results are expressed in nanogram of HIF-1α and picogram of VEGF per milliliter of cell lysis

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medium.

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2.7. Preparation and characterization of injectable hydrogels

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SA from brown algae (SA; low viscosity) and gluconic acid δ–lactone (GDL; > 99.0%) were

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purchased from Sigma-Aldrich. Calcium chloride (CaCl2) was obtained from Sinopharm

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Chemical Reagent Co., Ltd. (Shanghai, China).The composite hydrogels were fabricated by a

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similar method as previously reported33,37. According to our previous work42, SA hydrogels

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containing 0.02 g/mL of silicate ceramics, such as akermanite, exhibit excellent injectability and

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capacity to promote angiogenesis of HUVECs. In addition, our preliminary experiments showed

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that, after SA-BG hydrogels were soaked in simulated body fluid (SBF) for 24 hour, the

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concentration of released Si ion was about 40 folds of the optimal Si ion concentration for the

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angiogenesis of HUVECs in vitro. In order to ensure the ratio of DFO and Si ion concentrations

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and not to affect their synergistic effect in biological function, we designed our SA hydrogels as

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follows: for the BG containing SA hydrogel (denoted as SA-BG), 0.02 g of BG powders were

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added into a 1 mL of SA solution (2 wt. %) with 0.01 g of GDL followed by repeatedly pipetting

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in syringe to generate homogeneous solution. Under the weak acid condition of GDL, both of Ca

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and Si ions could be released from BG powders, and the released Ca ions contributed to the

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cross-linking of hydrogels. The DFO containing hydrogel (denoted as SA-DFO) was prepared by

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extruding 1 mL of SA solution containing 0.00056 g of DFO into CaCl2 solution (0.1 M) with

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needle-free syringe to crosslink for 55 seconds, followed by washing with deionized water for one

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time to remove the redundant CaCl2. The BG and DFO containing hydrogel (denoted as

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SA-BG/DFO) was prepared by a similar process with SA-BG with additional 0.00056 g of DFO.

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Finally, the obtained composite hydrogels were extruded for further characterization and

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utilization.

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To characterize the fabricated hydrogels, hydrogels were first observed with a field emission



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scanning electron microscope (SEM; S-4800, Hitachi, Japan). The gelling time of hydrogels was

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defined as the period of time from the initial addition of BG powders in SA solution to the

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stopped-flow of hydrogel according to previous publication and recorded with a stopwatch33. Then,

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their water content and swelling ratio were measured according to our reported work33. In details,

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the wet weight (just prepared state, Wwet) and the dry weight (after the hydrogels were dried at

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37 °C thoroughly, Wdry) of the constructed hydrogels were measured with electronic scales, while

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the dry volume (after the hydrogels were dried at 37 °C thoroughly, Vdry) and the swelling volume

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(after the dry hydrogels were immersed in SBF at 37 °C for 24 hours, Vswelling) of the constructed

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hydrogels were measured through drainage method. Afterwards, their water content was

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calculated by: water content (%) = (Wwet – Wdry) / Wdry × 100%; their swelling ratio was defined

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as: swelling ration (%) = (V – Vdry) / Vdry × 100%.

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For their extracts, 1 mL of hydrogels was immersed in 10 mL of simulated body fluid (SBF)

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at 37 °C for 24 h, and the concentration of Ca, P, Si and DFO in supernatant was detected by

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ICP-AES and UV-visible spectroscopy (UV-visible 8500 spectrophotometer), respectively. To

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investigate the DFO release behavior of the DFO containing composite hydrogels, 1 mL of

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SA-DFO or SA-BG/DFO was soaked in 10 mL of SBF buffer and shaken in a humidified shaking

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table at 37 °C at a mild speed. After 2 ~ 72 hours, 1 mL of supernatants were collected (1 mL of

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fresh SBF was supplemented to the immersion system immediately) and the concentration of DFO

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in each supernatant was measured by UV-visible spectroscopy at 485 nm wavelength12. Finally,

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the results were reflected as cumulative release percentage.

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2.8. Induction of diabetes and excisional wound splinting model preparation

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All protocols obtained approval from the Animal Care and Experimental Committee of Sixth

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People's Hospital affiliated to Shanghai Jiao Tong University School of Medicine. Sixty male

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Sprague-Dawley rats aged 8 weeks were selected and observed for one week before diabetes

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induction. All rats were fasted for a night before diabetes induction and the baseline blood glucose

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level was measured. The diabetic model was built using streptozotocin (65 mg/kg b.w., i.p.) the

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next morning. The blood glucose level was measured again three days later. Only rats with a blood

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glucose level above 300 mg/dL were chosen as the experimental rats. Before preparing skin

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wounds, the rats were observed for another two weeks.

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Forty-eight rats with a blood glucose level above 300mg/dL were randomly selected for the


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experiment. The excisional wound splinting model was established based on the previous

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method43. The wound diameter was 20mm. For in vivo investigations of hydrogels, SA, BG

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powders and DFO were first sterilized by being adequately exposed under ultra-violet light, while

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the deionized water was sterilized through high temperature at 121 °C for 30 min, and all the

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composite hydrogels were prepared in a sterile environment. The prepared SA-BG, SA-DFO and

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SA-BG/DFO hydrogels were respectively injected into the wounds during surgery. After the

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hydrogels solidified, the wound surface was dressed with gauze and bandage. We adopted the

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operation method of replacing a new hydrogel dressing every 3 days according to the dressing

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change method in clinical practice. This operation method can also ensure effective DFO

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concentration in wound site during the 20 days.

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2.9. Wound closure measurement and histological analysis

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Wound healing process was captured with a digital camera at day 0, day 5, day 12 and day 20,

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respectively. Wound area was calculated using the image analysis software (NIH Image). Wound

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closure was calculated as below: %wound closure= (A0-At)/ A0 ×100, where A0 is the wound area

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at day 0, and At is the wound area at day 5, day 12 and day 20, respectively.

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Rats were sacrificed at day 12 and day 20, respectively. Skin was sampled from the wound

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surface and at 5mm from the wound surface. The skin samples were fixed in 10% formalin,

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dehydrated in gradient alcohol and embedded in paraffin. Then, the specimens were cut into 5 µm

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sections. Hematoxylin-eosin (H&E) and Masson's trichrome staining were carried out. The

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equation below was used to calculate the wound reepithelializaton rate (E%): E% = LN/LO × 100%.

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In the equation, LO indicates the primitive wound length, and LN indicates the length of new

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epithelium. Image J Software was used to determine the proportion of collagen deposition by

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measuring the intensity of the blue areas according to the previous study2-3. To assess the effect of

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BG and DFO on the secretion of HIF-1α and VEGF, immunohistochemical (IHC) method was

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used to detect HIF-1α and VEGF. In addition, The IHC assessment for HIF-1α and VEGF was

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based on the staining intensity and stained proportion, and five random fields at a 400 ×

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magnification were evaluated. The quantification of IHC was counted according to the previous

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study44. To assess vascular system formation, immunohistochemical staining was conducted for

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CD31 (Abcam, Great Britain; 1:200), and immunofluorescence (IF) staining was carried out for

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CD31 +α-smooth actin (α-SMA) (Abcam, Great Britain; 1:50). The number of blood vessels was



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counted blindly by two pathologists.

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2.10. Statistical analysis

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The mean ± standard deviation (SD) was used to describe the data, and SPSS software was

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employed for data analysis. Student–Newman–Keuls post hoc test and One-way analysis of

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variance were employed. p< 0.05 indicated significant difference.

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3. Results

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3.1. The effects of DFO concentration on cell proliferation, migration and tube

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formation

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Relationship between DFO concentration and HUVECs proliferation, migration and tube

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formation are shown in Figure 1. It can be seen that, compared with the control group, DFO of 8

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µM and above showed significant cytotoxicity on HUVECs, while DFO of 4µM and below had no

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significant impact on HUVECs. Therefore, DFO of 4µM and below was selected for the

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subsequent migration assay and tube formation assay. Figure 1B and D indicate that, compared

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with the control group, addition of DFO in the cell culture medium significantly improved the

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number of migrating cells. Among DFO concentration ranging from 4 to 0.5 µM, 2 µM DFO

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showed the highest stimulatory effects on HUVECs migration. Similarly, the tube formation

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capacity of HUVECs was significantly enhanced by addition of DFO as compared with the control

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group and 2 µM DFO showed the highest stimulatory effects on HUVECs tube formation among

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all groups (Figure 1C and E). It was then concluded that 2 µM DFO was not only cytocompatible

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but also possessed the highest stimulatory effects on migration and tube formation of HUVECs.

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So, 2 µM DFO was chosen for the subsequent in vitro experiments.


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Figure 1. Effects of DFO on proliferation, migration and tube formation of HUVECs. (A) CCK-8

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assay. (B) Transwell migration. (C) Tube formation. (D) Quantitation of cell migration (violet

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stained cells). (E) Quantitative evaluation of tube formation. (* indicates significant differences

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between control and 0.5-16 µM groups; % indicates significant differences between 0.5 µM and

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1-4 µM groups; # indicates significant differences between 1 µM and 2-4 µM groups; & indicates

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significant differences between 2 µM and 4 µM group. p< 0.05).

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3.2. The effects of DFO, BG ionic products and combination of DFO and BG ionic products on cell proliferation, migration and tube formation

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CCK-8 assay was performed again to determine the cytocompatibility of the BG ion extract

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(Si ion concentration, 1 ug/mL), 2µM DFO and combination of 1 µg/mL BG and 2 µM DFO. The

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results are shown in Figure 2A. It can be seen that neither BG ion extract nor BG+DFO



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combination had apparent cytotoxicity on HUVECs and cell culture media either containing 1

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µg/mL Si ions of BG ionic products, 2 µM DFO or combination of 1 µg/mL Si ions and 2 µM

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DFO showed the same effects on HUVECs proliferation. According to migration assay (Figure 2B

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and D) and tube formation assay (Figure 2C and E), BG+DFO combination showed the strongest

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promoting effects on migration and tube formation capacities of HUVECs, as compared with the

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control group, BG group and DFO groups. This indicated that BG and DFO worked

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synergistically in promoting the migration and tube formation of HUVECs.

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Figure 2. Effects of DFO, BG and combination of DFO and BG on proliferation, migration and

10

tube formation of HUVECs (A) CCK-8 assay. (B) Transwell migration. (C) Tube formation. (D)

11

Quantitation of cell migration (violet stained cells). (E) Quantitative evaluation of tube formation


Page 12 of 28

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1

(* indicates significant differences between control and BG; DFO or BG+DFO groups; %

2

indicates significant differences between BG and DFO or BG+DFO groups; # indicates significant

3

differences between DFO and BG+DFO groups. p < 0.05).

4 5

3.3. The effects of DFO, BG ionic products and combination of DFO and BG ionic

6

products on vascularization growth factors

7

As compared with the control group where HUVECs were cultured with ECM, addition of

8

BG ionic products, DFO and combination of BG ionic products and DFO significantly stimulated

9

the gene expressions of HIF-1α (Figure 3A) and VEGF (Figure 3B). In addition, DFO and

10

BG+DFO groups showed higher stimulatory effects on HIF-1α gene expression in HUVECs than

11

BG while there was no significant difference between DFO and BG+DFO groups in terms of

12

stimulating HIF-1α gene expression in HUVECs. Addition of BG ionic products, DFO and

13

combination of BG ionic products and DFO also significantly stimulated the gene expressions of

14

VEGF as compared with the control group where HUVECs were cultured with ECM. There was

15

no significant difference in the gene expressions of VEGF in HUVECs cultured with BG or DFO.

16

However, it is interesting to note that the combination of BG ionic products and DFO (BG+DFO

17

group) showed considerably higher stimulatory effects on gene expressions of VEGF than BG and

18

DFO groups. This was especially true in the expression of VEGF gene as the VEGF gene

19

expression in HUVECs cultured with BG+DFO was almost two times higher than that in

20

HUVECs cultured with BG or DFO.

21

HIF-1α and VEGF protein expressions are shown in Figure 3C and D, which were consistent

22

with gene expression results. The secretion of HIF-1α and VEGF was promoted in the BG, DFO

23

and BG+DFO groups as compared to that in the control group. Among these three groups,

24

BG+DFO group showed the highest stimulatory effects and BG group showed the lowest

25

stimulatory effects on HIF-1α expression in HUVECs (Figure 3C). As to VEGF expression, there

26

was no significant difference between the BG group and DFO group while the expression level

27

was higher in the HUVECs cultured with BG+DFO than in those cultured with either BG or DFO

28

(Figure 3D).



1 2

Figure 3. Effects of BG, DFO and BG+DFO on HIF-1α and VEGF expression and

3

characterization of injectable hydrogels. (A and B) qRT-PCR analysis of HIF-1α and VEGF

4

mRNA in HUVECs. (C, D) Expression of HIF-1α and VEGF detected by ELISA. (E) SEM

5

images of porous structure of hydrogels. (F) Concentrations of ions in SBF, released from SA-BG

6

and SA-BG/DFO hydrogels after the hydrogels were soaked in SBF at 37℃ for 24 hours. (G)

7

Profiles of DFO released from SA-BG/DFO and SA-DFO composites. (*indicates significant

8

differences between control and BG, DFO or BG+DFO groups; % indicates significant differences

9

between BG and DFO or BG+DFO groups; # indicates significant differences between DFO and

10

BG+DFO groups. p< 0.05).

11 12

3.4. Characterization of different hydrogels and DFO release behavior

13

As shown in Figure 3E, the SEM images display that both SA-BG and SA-BG/DFO possess

14

relatively smooth surface and interconnecting porous structure without obvious difference. In

15

Figure 3F, clearly, there is no remarkable difference between SA-BG and SA-BG/DFO extracts in

16

the concentration of Si ions, which is 39.0 and 39.6 µg/mL, respectively. Therefore, addition of

17

DFO in the SA-BG/DFO hydrogels did not change the ion release behavior of BG from the

18

hydrogels. These concentrations are about 40 times higher than the effect concentration of Si ion

19

(1 µg/mL).Water content, swelling ratio and gelling time of the hydrogels were shown in the

20

supporting information (Figure S1).

21

Regarding the DFO release behavior, Figure 3G indicates that there is no significant

22

difference between the DFO release rate in SA-DFO and SA-BG/DFO hydrogels. Therefore,

23

addition of BG in the SA-DFO did not change the DFO release behavior from the hydrogels.


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1

Within the first 12 hours after the soaking of composite hydrogels in SBF buffer, DFO was

2

released linearly. After 24 hours, about 80% of DFO has been released and the final cumulative

3

release percentages reach to 87~89.5%.

4

3.5. Gross observation of skin wound and wound closure calculation

5

Figure 4A shows the images of diabetic wounds treated with different materials for different

6

periods. None of the wound surface had apparent infection at each stage after surgery and the

7

wound area decreased over time. The wound repair was the fastest in the SA-BG/DFO group, and

8

the newly formed skin had basically covered the wound surface at day 20. Figure 4C shows the

9

wound closure in each group. It can be seen that the SA-BG group and SA-DFO group had a

10

much higher rate of wound closure as compared with the control group and that the two groups

11

did not differ significantly. In addition, compared with the SA-BG group and SA-DFO group, the

12

SA-BG/DFO group showed a more rapid wound closure.

13 14

Figure 4. Gross observation of wound healing in an excisional diabetic wound splinting model.

15

(A)Cutaneous defects images of wounds treated with nothing (control), SA-BG hydrogels

16

(SA-BG), SA-DFO composites (SA-DFO), SA-BG/DFO hydrogels (SA-BG/DFO) at 0, 5, 12 and

17

20 days post-operation. (B) Photos of SA-BG/DFO composite hydrogels. (C) Wound closure rate

18

of the defects treated with different kinds of materials. (* indicates significant differences between

19

control and SA-BG, SA-DFO or SA-BG/DFO groups; % indicates significant differences between



1

SA-BG and SA-DFO or SA-BG/DFO DFO groups; # indicates significant differences between

2

SA-DFO and SA-BG/DFO groups. p < 0.05).

3 4

3.6. Histologic, immunohistochemical and immunofluorescent evaluation

5

H&E and Masson’s staining were performed on the samples taken from the wound sites to

6

assess the repair of diabetic wound. Figure 5 shows the H&E stained images (Figure 5A) and

7

reepithelialization rate statistical analysis (Figure 5B). It can be seen that reepithelialization rates

8

were the highest in the SA-BG/DFO group at day 12 and day 20, reaching 62% and 92.6%,

9

respectively, which were much higher as compared with those in the control, SA-BG and SA-DFO

10

group (41.3%, 60.3%;55.7%, 80.3%; 51.3%, 77%) (Figure 5B).

11 12

Figure 5. H&E staining of wound sections obtaining from control, SA-BG, SA-DFO or

13

SA-BG/DFO groups at 12 and 20 days post-operation. The arrows indicate the edges of the

14

wounds. (A) H&E stained images (B) Wound reepithelialization rate of the defects treated with

15

different kinds of materials at 12 and 20 days post-operation (* indicates significant differences

16

between control and SA-BG, SA-DFO or SA-BG/DFO groups; % indicates significant differences

17

between SA-BG and SA-DFO or SA-BG/DFO DFO groups; # indicates significant differences

18

between SA-DFO and SA-BG/DFO groups. p < 0.05).

19 20

Collagen deposition and maturation were further assessed in each group by Masson’s

21

trichrome staining. Figure 6 shows the Masson’s trichrome stained images (Figure 6A) and


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1

collagen proportion statistical analysis (Figure 6B). It can be seen that the SA-BG/DFO group not

2

only had the highest collagen deposition reflected by the deep blue staining, but also the most

3

orderly collagen arrangement compared with other groups at both 12 and 20 days. In addition,

4

more mature fibers with regular orientation and distribution were shown in the wound sites treated

5

with SA-BG/DFO as compared to those treated with SA-DFO, SA-BG hydrogels or nothing

6

(control).

7 8

Figure 6. Masson's trichrome staining of wound sections obtaining from control, SA-BG,

9

SA-DFO or SA-BG/DFO groups at 12 and 20 days post-operation, showing collagen deposition

10

and maturity. (A) Masson’s trichrome stained images. (B) The Image J software was used to

11

quantify the collagen deposition in the wound sites. (* indicates significant differences between

12

control and SA-BG, SA-DFO or SA-BG/DFO groups. p < 0.05).

13 14 15

Figure 7 shows the IHC stained images (Figure 7A and B) and IHC evaluation statistical

16

analysis (Figure 7C and D) of HIF-1α and VEGF expression. At day 12, the staining of HIF-1α

17

and VEGF was the deepest in the SA-BG/DFO group, indicating the highest expressions of

18

HIF-1α and VEGF in this group among all groups. This further confirmed the results from in vitro

19

experiment that combined use of BG and DFO led to the highest expressions of HIF-1α and

20

VEGF than the use of either alone. However, at day 20, the intensity of staining was similar across

21

the groups without significant difference.



1 2

Figure 7. IHC analysis of angiogenic factor expression in wound sites of control, SA-BG,

3

SA-DFO or SA-BG/DFO groups. (A) IHC staining for HIF-1α. (B) IHC staining for VEGF. (C)

4

Quantification of IHC staining of HIF-1α. (D) Quantification of IHC staining of VEGF. (*

5

indicates significant differences between control and SA-BG, SA-DFO or SA-BG/DFO groups; %

6

indicates significant differences between SA-BG and SA-DFO or SA-BG/DFO DFO groups; #

7

indicates significant differences between SA-DFO and SA-BG/DFO groups. p < 0.05).

8 9

CD31 immunohistochemical staining was conducted to show the new blood vessels

10

formation, and CD31/α-SMA double immunofluorescence staining was performed to indicate the

11

mature blood vessels. Figure 8A and C show CD31 staining and Figure 8B and D show

12

CD31+α-SMA double staining, respectively. At day 12, the number of newly formed and mature

13

blood vessels was higher in the SA-BG, SA-DFO and SA-BG/DFO groups than that in the control

14

group. The highest number of newly formed and mature blood vessels was observed in the

15

SA-BG/DFO group. However, at day 20, the control group had the highest number of newly

16

formed and mature blood vessels. The reduction in the number of newly formed and mature blood

17

vessels was greatest in the SA-BG/DFO group at day 20 though the number of newly formed and

18

mature blood vessels of this group was the highest at day 12.


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1 2

Figure 8. IHC and IF analysis of neovascularization in wound sites of control, SA-BG, SA-DFO

3

or SA-BG/DFO groups. (A) IHC staining for CD31. (B) IF co-staining for a-SMA and CD31. (C)

4

Quantitative analysis of newly formed blood vessels at 12 and 20 days post-operation. (D)

5

Quantitative analysis of mature vessels at 12 and 20 days post-operation. (* indicates significant

6

differences between control and SA-BG, SA-DFO or SA-BG/DFO groups; % indicates significant

7

differences between SA-BG and SA-DFO or SA-BG/DFO groups; # indicates significant

8

differences between SA-DFO and SA-BG/DFO groups. p < 0.05).

9

4. Discussion

10

Diabetic wound healing remains a clinical challenge. Skin wound repair is a complex process

11

involving multiple factors and cells, all of which are related to angiogenesis. Although many

12

attempts have been conducted by researchers, such as single use of drugs or bioactive materials to

13

improve angiogenesis during wound healing, the outcomes are unsatisfactory. In this study, it was

14

the first time to combine the drugs and bioactive materials to promote angiogenesis, and a novel

15

hydrogel containing both DFO and BG was prepared. Through in vitro experiments, we first found

16

that DFO concentration of 2 µM was most favorable for endothelial cell migration and tube

17

formation. The combination of DFO and BG at their optimal concentrations achieved the best

18

effect in promoting HUVECs migration and tube formation in vitro than the use of either BG or

19

DFO alone.

20

The complex formed by HIF-1α and p300, through binding to hypoxia response elements,



1

can up-regulate expression of several angiogenic genes, such as stromal cell-derived factor 1 and

2

VEGF45-46. Its ability in greatly upregulating the expressions of VEGF and SDF-1α has been

3

widely reported12. In this study, we found that DFO was more potent than BG in promoting the

4

expression of HIF-1α. DFO is a ferric ion chelator used clinically to treat acute iron poisoning. In

5

normoxic conditions, DFO can be chelated with ferric ions, thus reducing the degradation of

6

HIF-1α and promoting its expression. Thus, DFO acts as a hypoxia mimetic agent in normoxia47-48.

7

In our previous study and other studies, it has reported that Si ions inhibit the degradation of

8

HIF-1α and mildly upregulate the expression of HIF-1α by stabilizing it24,49-50. As has been

9

indicated by many researches, BG can promote angiogenesis51-53, but the working mechanism

10

remains unclear. Neither has it been clarified that BG promotes angiogenesis by upregulating the

11

expression of HIF-1α in cells. According to our in vitro experiments, BG only mildly increased the

12

expression of HIF-1α and the degree of increase was lower compared to that of DFO did. It is

13

interesting to note that the combined use of DFO and BG further increased the expressions of

14

VEGF. A synergistic effect was observed between the two in the upregulation of VEGF expression.

15

It has already been confirmed that DFO upregulates the expression of HIF-1α by mimicking a

16

hypoxic environment, thereby upregulating VEGF12,54-55. BG, however, promotes intercellular

17

communication which will increase the expression of VEGF by paracrine regulation and

18

moderation of gap junctional intercellular communication21,38,56-58. This may be the underlying

19

explanation for the synergistic effect between BG and DFO in promoting the upregulation of

20

VEGF. More evidences are needed to support this hypothesis, and the existing studies have

21

demonstrated the key roles played by VEGF in revascularization. We have shown for the first time

22

that drugs and biomaterials, such as ionic products, also have a synergistic effect in promoting the

23

expression of certain growth factors probably through different signal pathways.

24

Based on the in vitro cell experiments, we developed an injectable hydrogel containing both

25

BG and DFO on the basis of previous studies. The results showed that the hydrogel containing

26

only BG was not significantly different from that containing both BG and DFO in the amount of

27

Ca and Si ions released within 24 hours. That means DFO does not interfere with the release of

28

ionic products from BG. In addition, compared with hydrogel containing both BG and DFO, the

29

hydrogel containing only DFO was not significantly different in terms of the release rate of DFO.

30

Both could achieve a sustained release within 3 days, a normal interval for dressing changes in


Page 20 of 28

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1

clinical practice. This demonstrates the clinical application potential of this hydrogel. The

2

hydrogel prepared in this study was in a liquid state at the beginning and injected to the site of

3

skin injury with a syringe. After entering the wound, BG will be dissolved in the weakly acidic

4

environment created by GDL to give Ca ions that crosslink with SA. As a result, a gel is formed

5

over the wound and lasts for 45 ~ 75 seconds. Before solidification, the hydrogel can fully cover

6

the wound. The water content of the hydrogel after solidification is over 90%, which creates a

7

moist environment favorable for wound repair2.

8

In the animal experiment, tissues surrounding the wound surface were immobilized with a

9

steel loop, which reduced the influence of skin shrinkage on wound closure. Therefore, wound

10

closure will be achieved completely through granulation tissues and epidermal regeneration. As a

11

result, the rat model of skin wound repair would more resemble the actual wound repair in clinic59.

12

Hydrogel was then applied to the diabetic rat skin defect model. At day 5, day 12 and day 20, the

13

hydrogel containing BG or DFO achieved faster wound closure as compared with the control

14

group, but there is no statistical significance between the two groups. The SA-BG/DFO group had

15

an even greater speed of wound closure than the SA-BG group and SA-DFO group. A dry

16

environment on the outside will inhibit epidermal regeneration, while a moist environment is

17

conducive to it36,60. A high water content of hydrogel (over 90%) ensures a moist environment for

18

wound repair. In addition, the hydrogen will continuously release Ca and Si ions of effective

19

concentrations to promote the expressions of VEGF and HIF-1α and hence revascularization. In

20

the meantime, DFO, as another ingredient, is also released continuously from hydrogel into the

21

wound surface within 3 days to promote the expressions of VEGF and HIF-1α and

22

revascularization. This is further conducive to the formation of granulation tissues and collagen

23

deposition61.

24

Healthy granulation tissues are also important for epidermal regeneration by supplying

25

nutrients and growth factors necessary for epidermal regeneration. This is especially important for

26

chronic ischemic wound surface. As found by H&E staining, the epidermal regeneration rate was

27

consistent with gross appearance to naked eyes in each group, both indicating a trend towards

28

wound healing. A gradual replacement of granulation tissues by collagen fibers is a basic process

29

of skin wound repair. By Masson’s staining we observed that more collagen was deposited in the

30

wound surface in the SA-BG, SA-DFO and SA-BG/DFO group than the control group and the



1

collagen fibers were more orderly arranged. The highest amount and the most orderly arrangement

2

of deposited collagen fibers were observed in the BG+DFO group, and they are very much

3

resembled the normal skin structure.

4

From the in vivo experiment, we verified the findings from the in vitro experiment by

5

immunochemical staining for HIF-1α and VEGF. That is, BG and DFO greatly promoted the

6

expressions of HIF-1α and VEGF and they worked synergistically, especially in the case of VEGF,

7

a crucial growth factor in revascularization62. It has been generally recognized that VEGF

8

promotes revascularization in skin wound repair, consistent with the staining for CD31/α-SMA in

9

our study. At day 12, the number of newly formed and mature blood vessels was higher in the

10

SA-BG, SA-DFO and SA-BG/DFO groups as compared with the control, and the highest was

11

observed in the SA-BG/DFO group. At day 20, the number of newly formed and mature vessels

12

was the highest in the control group while the number of newly formed and mature vessels in

13

SA-BG/DFO group decreased at day 20 while the wounds in control group still were in repair

14

progress. In normal dermal tissue, there are fewer blood vessels63. However, when the skin is

15

damaged, proper inflammatory reaction induces angiogenesis to provide oxygen and nutrient6,64

16

for cell and tissue activities in proliferation phase of wound healing. In this study, at day 12, the

17

number of newly formed and mature blood vessels was highest in the SA-BG/DFO groups among

18

all groups, indicating that SA-BG/DFO could stimulate blood vessel formation and facilitated skin

19

regeneration in proliferation phase. At day 20, SA-BG/DFO group had almost completed the

20

proliferation stage (as shown in Figure 5) and moved to the extracellular matrix remodeling phase.

21

In this phase, small blood vessels would gradually shrink and disappear, leaving only a small part

22

of the necessary vascular tissue. In contrast, the control group delayed tissue regeneration because

23

it could not promote angiogenesis in the early stage. At day 20, tissue regeneration in control

24

group was still not completed and a large number of blood vessels were still needed. So, there

25

were more blood vessels in this group than SA-BG/DFO groups. All these phenomena suggest that

26

the SA-BG/DFO materials can promote skin regeneration. Thus, revascularization started earliest

27

in the SA-BG/DFO group and the granulation tissues became mature gradually and the wound

28

surface healed fastest in the SA-BG/DFO group. Although BG and DFO work synergistically in

29

promoting VEGF expression and revascularization, the specific mechanism remains unknown. We

30

have demonstrated that the combined use of BG and DFO is more potent in inducing skin wound


Page 22 of 28

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1

repair than the use of either alone. This novel type of sheds light on the development of new

2

dressings for wound.

3

5. Conclusion

4

In this study, it was the first time to combine angiogenic drug DFO and bioactive material

5

BG to promote angiogenesis, and a novel hydrogel containing both DFO and BG was prepared.

6

DFO may induce hypoxia condition for cells and trigger the vascularization or angiogenesis while

7

BG can enhance vascularization mainly due to its ionic effects on stimulation of angiogenic

8

growth factor expression in endothelial cells. Our results demonstrated that the combination of BG

9

and DFO significantly stimulated migration and tube formation of HUVECs as compared with the

10

single use of BG or DFO alone, which indicates that BG and DFO could synergistically upregulate

11

gene expression of VEGF. Therefore, the injectable hydrogels containing both BG and DFO better

12

facilitated angiogenesis and diabetic wound healing than the use of BG or DFO alone. All these

13

results indicate that the combination of angiogenic drug and bioactive material, such as DFO and

14

BG, might be a promising way to enhance vascularization for wound healing and this method can

15

also be feasible for regenerating other vascularized tissues.

16 17 18 19

Conflicts of interest The authors have declared that no conflict of interest exists.

Acknowledgements

20

This work was supported by the National Key Research and Development Program of China

21

(Grant No. 2016YFC1100201), National Natural Science foundation of China (81572106,

22

31470918 and 31771024) and the Interdisciplinary Program of Shanghai Jiao Tong University

23

(YG2017MS20).

24

Supporting Information

25

Table S1. Gene sequence of primers used in this study.

26

Figure S1. Characterization of composite hydrogels.

27 28

References

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2. Zhao, L.; Niu, L.; Liang, H.; Tan, H.; Liu, C.; Zhu, F. Ph and Glucose Dual-Responsive Injectable Hydrogels with Insulin and Fibroblasts as Bioactive Dressings for Diabetic Wound Healing. ACS Appl Mater Interfaces 2017, 9 (43), 37563-37574. 3. Yoon, D. S.; Lee, Y.; Ryu, H. A.; Jang, Y.; Lee, K. M.; Choi, Y.; Choi, W. J.; Lee, M.; Park, K. M.; Park, K. D.; Lee, J. W. Cell Recruiting Chemokine-Loaded Sprayable Gelatin Hydrogel Dressings for Diabetic Wound Healing. Acta Biomater 2016, 38, 59-68. 4. Li, J.; Chen, J.; Kirsner, R. Pathophysiology of Acute Wound Healing. Clin Dermatol 2007, 25 (1), 9-18. 5. Telgenhoff, D.; Shroot, B. Cellular Senescence Mechanisms in Chronic Wound Healing. Cell Death Differ 2005, 12 (7), 695-698. 6. Mandriota, S. J.; Pepper, M. S. Vascular Endothelial Growth Factor-Induced in Vitro Angiogenesis and Plasminogen Activator Expression Are Dependent on Endogenous Basic Fibroblast Growth Factor. J Cell Sci 1997, 110 ( Pt 18), 2293-2302. 7. Rocha, F. G.; Sundback, C. A.; Krebs, N. J.; Leach, J. K.; Mooney, D. J.; Ashley, S. W.; Vacanti, J. P.; Whang, E. E. The Effect of Sustained Delivery of Vascular Endothelial Growth Factor on Angiogenesis in Tissue-Engineered Intestine. Biomaterials 2008, 29 (19), 2884-2890. 8. Kanczler, J. M.; Ginty, P. J.; Barry, J. J.; Clarke, N. M.; Howdle, S. M.; Shakesheff, K. M.; Oreffo, R. O. The Effect of Mesenchymal Populations and Vascular Endothelial Growth Factor Delivered from Biodegradable Polymer Scaffolds on Bone Formation. Biomaterials 2008, 29 (12), 1892-1900. 9. O'Neill, H. S.; Herron, C. C.; Hastings, C. L.; Deckers, R.; Lopez Noriega, A.; Kelly, H. M.; Hennink, W. E.; McDonnell, C. O.; O'Brien, F. J.; Ruiz-Hernandez, E.; Duffy, G. P. A Stimuli Responsive Liposome Loaded Hydrogel Provides Flexible on-Demand Release of Therapeutic Agents. Acta Biomater 2017, 48, 110-119. 10. Yao, Q.; Liu, Y.; Tao, J.; Baumgarten, K. M.; Sun, H. Hypoxia-Mimicking Nanofibrous Scaffolds Promote Endogenous Bone Regeneration. ACS Appl Mater Interfaces 2016, 8 (47), 32450-32459. 11. Drager, J.; Sheikh, Z.; Zhang, Y. L.; Harvey, E. J.; Barralet, J. E. Local Delivery of Iron Chelators Reduces in Vivo Remodeling of a Calcium Phosphate Bone Graft Substitute. Acta Biomater 2016, 42, 411-419. 12. Chen, H.; Jia, P.; Kang, H.; Zhang, H.; Liu, Y.; Yang, P.; Yan, Y.; Zuo, G.; Guo, L.; Jiang, M.; Qi, J.; Liu, Y.; Cui, W.; Santos, H. A.; Deng, L. Upregulating Hif-1alpha by Hydrogel Nanofibrous Scaffolds for Rapidly Recruiting Angiogenesis Relative Cells in Diabetic Wound. Adv Healthc Mater 2016, 5 (8), 907-918. 13. Duscher, D.; Neofytou, E.; Wong, V. W.; Maan, Z. N.; Rennert, R. C.; Inayathullah, M.; Januszyk, M.; Rodrigues, M.; Malkovskiy, A. V.; Whitmore, A. J.; Walmsley, G. G.; Galvez, M. G.; Whittam, A. J.; Brownlee, M.; Rajadas, J.; Gurtner, G. C. Transdermal Deferoxamine Prevents Pressure-Induced Diabetic Ulcers. Proc Natl Acad Sci U S A 2015, 112 (1), 94-99. 14. Duscher, D.; Januszyk, M.; Maan, Z. N.; Whittam, A. J.; Hu, M. S.; Walmsley, G. G.; Dong, Y.; Khong, S. M.; Longaker, M. T.; Gurtner, G. C. Comparison of the Hydroxylase Inhibitor Dimethyloxalylglycine and the Iron Chelator Deferoxamine in Diabetic and Aged Wound Healing. Plast Reconstr Surg 2017, 139 (3), 695e-706e. 15. Hench, L. L.; Splinter, R. J.; Allen, W. C.; Greenlee, T. K. Bonding Mechanisms at the Interface of Ceramic Prosthetic Materials. J Biomed Mater Res 1971, 5 (6), 117–141. 16. Wilson, J.; Low, S. B. Bioactive Ceramics for Periodontal Treatment: Comparative Studies in the Patus Monkey. J Appl Biomater 1992, 3 (2), 123-129.


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TOC 251x176mm (300 x 300 DPI)


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Bioactive Injectable Hydrogels Containing Desferrioxamine and

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