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Composite hydrogel modified by IGF-1C domain improves stem cell therapy for limb ischemia Xiaomin Wang, Jimin Zhang, Weilong Cui, Yuan Fang, Li Li, Shenglu Ji, Duo Mao, Tingyu Ke, Xin Yao, Dan Ding, Guowei Feng, and Deling Kong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b17533 • Publication Date (Web): 12 Jan 2018 Downloaded from http://pubs.acs.org on January 15, 2018

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Composite Hydrogel Modified by IGF-1C Domain Improves Stem Cell Therapy for Limb Ischemia Xiaomin Wanga#, Jimin Zhanga#, Weilong Cuia, Yuan Fanga, Li Lia, b, Shenglu Jia, Duo Maoa, Tingyu Keb, Xin Yaoc, Dan Dinga, Guowei Fengc, *, Deling Konga, *

a

State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive

Materials, Ministry of Education, College of Life Science, Nankai University, Tianjin 300071, China b

Department of Endocrinology, The Second Affiliated Hospital, Kunming Medical

University, Kunming 650101, Yunnan, China c

Department of Genitourinary Oncology, Tianjin Medical University Cancer Institute

and Hospital, National Clinical Research Center for Cancer, Tianjin Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin 300060, China

Corresponding Authors *Deling Kong: [email protected]. *Guowei Feng: [email protected]

#

Xiaomin Wang and Jimin Zhang contributed equally to this work.

KEYWORDS: hydrogel, insulin-like growth factor 1 (IGF-1), adipose-derived stromal cells (ADSCs), critical limb ischemia (CLI), neovascularization

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ABSTRACT Stem cell treatment for critical limb ischemia yields a limited therapeutic effect due to cell loss and dysfunction caused by local ischemic environment. Biomimetic scaffolds emerge as ideal cell delivery vehicles for regulating cell fate via mimicking the components of stem cell niche. Herein, we prepared a bioactive hydrogel by mixing chitosan and hyaluronic acid that is immobilized with C domain peptide of IGF-1 (IGF-1C), and examined whether this hydrogel could augment stem cell survival and therapeutic potential. Our results showed that IGF-1C-modified hydrogel increased in vitro viability and pro-angiogenic activity of adipose-derived stromal cells (ADSCs). Moreover, co-transplantation of hydrogel and ADSCs into ischemic hindlimbs of mice effectively ameliorated blood perfusion and muscle regeneration, leading to superior limb salvage. These therapeutic effects can be ascribed to improved ADSC retention, angiopoientin-1 secretion and neovascularization, as well as reduced inflammatory cell infiltration. Additionally, hydrogel enhanced anti-fibrotic activity of ADSCs as evidenced by decreased collagen accumulation at late stage. Together, our findings indicate that composite hydrogel modified by IGF-1C could promote survival and pro-angiogenic capacity of ADSCs and thereby represents a feasible option for cell-based treatment for critical limb ischemia.

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1. INTRODUCTION Critical limb ischemia (CLI) is the most severe form of peripheral artery disease with high disability and mortality1-2. Loss of vascular supply is the main pathophysiologic feature of CLI, and leads to tissue infection, ulcer, death, or gangrene. Revascularization is critical for patients with CLI to restore blood flow in the affected limb and reduce the risk of amputation3. However, up to 50% of patients are ineligible for conventional revascularization approaches. Thus, development of alternative therapeutic strategies is urgently needed to treat this intractable disease. Stem cells are rapidly emerging as an attractive candidate for CLI treatment. Increasing evidence from animal studies suggests that stem cells target multiple pathogenetic mechanisms and induce angiogenesis to support vascular regeneration4-5. For instance, local transplantation of adipose-derived stromal cells (ADSCs) stimulates blood vessel formation and protects ischemic tissues from necrosis via secretion of angiogenic growth factors6. However, the therapeutic efficacy of stem cells has been significantly impaired due to the fact that locally administrated cells are prone to undergo apoptosis in the ischemic7 and inflammatory8 environment. Bioscaffold-based tissue engineering strategies are being developed to enhance cell survival and function9-10. Evidence is accumulating to indicate that hydrogel can serve as a supportive niche for cell engraftment, which protects cells against inflammatory insult in ischemic tissue11. Chitosan (CS) and hyaluronic acid (HA) are both naturally derived polysaccharides that can form hydrogels with excellent biocompatibility and biodegradability12. A composite hydrogel based on CS and HA (CS-HA) was recently developed, in which CS and HA were incorporated to form a biodegradable, non-toxic injectable hydrogel without addition of a chemical crosslinking agent or radiant light13. The therapeutic benefits of CS-HA hydrogel have been identified in inflammatory and degenerative diseases, such as osteoarthritis14, periodontitis15, cartilage injury16, and disc defects17. In addition, this hydrogel was successfully used for loading a pro-angiogenic drug to enhance neovascularization18, indicating its potential use in treating ischemic disorders. Of note, several research efforts have illustrated that CS-HA scaffold provided an appropriate niche for encapsulated stem cells where the 3


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cell-matrix interaction and signaling may manipulate cell adhesion, proliferation, and differentiation19-20. However, to date there is no study demonstrating whether CS-HA hydrogel can affect the paracrine action and therapeutics of stem cells. Engineered microenvironments with hydrogel and functional proteins have shown promising results in regulating pro-angiogenic signaling of stem cells. For instance, extracellular matrix modified by fibronectin can influence secretion profile of stem cells, which leads to increased tubulogenesis of endothelial cells21 and enhanced wound healing22. What’s more, bioscaffold with muscle-mimicking geometrical cues can guide smooth muscle cell differentiation of stem cells23. Insulin-like growth factor 1 (IGF-1) is a strong mitogenic protein which contains 70 amino acids in a polypeptide chain composed of 4 domains24. IGF-1 can hasten skeletal muscle regeneration and protect cells against apoptosis25. Besides, it improves stem cell engraftment via activating phosphoinositide 3-kinase, which results in increased neovascularization26. The C domain of IGF-1 (IGF-1C) is recognized as functional region of its parental protein yet provides advantages in terms of sustained bioactivity, low cost, immune privilege, and the ease of synthesis. A proof-of-principle study has suggested that IGF-1C peptide had comparable beneficial effects to its full-sized protein in corneal wound healing27. Furthermore, we recently investigated the potential role of this bioactive peptide in mediating stem cell fate and found that IGF-1C-modified scaffold ameliorated cell survival and pro-angiogenic activity in a murine model of renal ischemia24. This implicates that designing a biomimetic matrix system by incorporating functional peptide might direct stem cell behavior by tuning cell-matrix interplay, and thus confer enhanced therapeutic effects. Hence, in this work we attempt to determine if an artificial niche modified by IGF-1 mimicking peptide could regulate therapeutic neovascularization of stem cells in ischemic limb. In the current work, we developed CS-HA-based composite hydrogel immobilized with IGF-1C peptide (CS-HA-IGF-1C), and hypothesized that this bioscaffold could enhance therapeutic potential of ADSCs in CLI via prolonging cell retention and regulating their paracrine actions. To test this hypothesis, we cultured ADSCs with the hydrogel and investigated the impacts of hydrogel on stem cell growth and 4



pro-angiogenic activity. Next, fluorescent probe-labeled ADSCs were transplanted with hydrogel into the ischemic limbs of mice to determine whether the hydrogel supported survival and therapeutics of ADSCs. Moreover, cell retention was monitored by molecular imaging and possible mechanism underlying accelerated muscle repair was also elucidated. 2. EXPERIMENTAL SECTION 2.1. IGF-1 Immobilization and Bioactive Scaffold Preparation. As illustrated in Figure 1B, the CS-HA-IGF-1C hydrogel was prepared by mixing succinyl-CS and IGF-1C conjugated aldehyde-HA. In brief, IGF-1C peptide was synthesized as documented in a previous study24. In parallel, HA (Tongze Biotech Co., Ltd, Xi’an, China) was oxidized by sodium periodate (Heowns Biochem Technologies LLC, Tianjin, China) to obtain aldehyde-HA as reported in the literature28. Then aldehyde-HA (10 mg/mL) was dissolved in PBS buffer (pH 7.4), and IGF-1C was added to aldehyde-HA at a feed molar ratio of 0.017. After that, IGF-1C modified aldehyde-HA (aldehyde-HA-IGF-1C) was purified by dialysis against a large excess amount of PBS for 1 day, and the desired product was obtained after being lyophilized. In the meantime, succinyl-CS was synthesized by adding succinic anhydride (Bailingwei China Chemical Co. Ltd, Beijing, China) to CS (deacetylation degree: 85%) (Haidebei Marine Bioengineering Co. Ltd, Jinan, China) as previously reported28. Afterwards, succinyl-CS (50 mg/mL) and aldehyde-HA-IGF-1C (35 mg/mL) were dissolved in PBS. Finally, the above two solutions were mixed at the ratio of 1:1 (v/v) to form the composite CS-HA-IGF-1C hydrogel via Schiff’s base linkage28. 2.2. Characterization of IGF-1C Modified Hydrogel. Fourier transformed infrared (FTIR) spectra of samples including CS, succinyl-CS, HA, aldehyde-HA and aldehyde-HA-IGF-1C were performed to confirm the expected pendant functionalities with an FTIR spectrometer (Bio-Rad). Rheological measurements of CS-HA scaffold and IGF-1C modified scaffold were carried out on rheometer (TA Instruments)24. Besides, the rheological properties of prepared scaffold samples were measured and 5


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the changes in modulus (G’ and G’’) were documented as functions of time. Furthermore, the morphology of IGF-1C modified hydrogel after lyophilization was examined using scanning electron microscopy (Quanta 200; FEI, Brno). 2.3. Cell Culture. For ADSC isolation, adipose tissues (peritoneal and inguinal site) were harvested from BALB/c mice. The isolated tissues were then digested and ADSCs were harvested, plated and expanded as previously reported24. ADSCs between passages 3 and 5 were used for experiments. In addition, human umbilical vein endothelial cells (HUVECs) were cultured using EGM-2 (Lonza). 2.4. Cell Cytotoxicity/Proliferation Assessment. The cytotoxicity of succinyl-CS and aldehyde-HA was investigated, respectively, by MTT assay using ADSCs29. ADSCs were seeded at a density of 10,000 cells per well. After 24 h, a series of concentration (0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 mg/mL) of succinyl-CS or aldehyde-HA were added into medium for another 24-h treatment. For proliferation assessment, ADSCs were encapsulated in 3D hydrogels and cultured in vitro. Briefly, ADSCs were suspended in CS-HA or CS-HA-IGF-1C pre-gelation solution. Then the hydrogels containing ADSCs were added into 48-well plate for completely covering the bottom of the well (100 µl per well). After gelation, the cell-hydrogel constructs were then cultured in α-MEM. The number of cells cultured in each hydrogel was evaluated using cell-counting kit-8 (CCK-8) (Dojindo Molecular Technologies) at different time points (24 h, 48 h, and 72 h). 2.5. Quantitative RT-PCR. To assess the impact of hydrogels on gene expression of ADSCs, cells suspended within CS-HA or CS-HA-IGF-1C pre-gelation solutions (1.0×106 cells in 300 µl hydrogel) were seeded into plates. For normal condition, ADSCs were cultured on plates for 3 days. After that, the cell-hydrogel constructs underwent incubation in α-MEM containing 5000 U hyaluronidase (Sigma) per 100 µl gel construct for 1 h. RNA isolation, cDNA synthesis, and quantitative PCR were carried out according to a previous study24. The sequences of primers used in the current work were listed in Table S1. 2.6. HUVEC Migration and Capillary Formation. To assess the impacts of hydrogels on paracrine actions of ADSCs, conditioned medium was collected from 6



the 3D-culture of ADSCs encapsulated in CS-HA or CS-HA-IGF-1C hydrogels (3.0×105 cells in 100 µl) in non-adherent 24-well plates. All medium samples collected from the plates after 48 h was mixed with EBM-2 (Lonza) (conditioned medium to EBM-2 ratio = 3:7) and was used for the treatment of HUVECs. To investigate the functional responses of HUVECs to paracrine signals from the encapsulated ADSCs, conditioned medium was added to the HUVEC culture. Then the migration and tube formation capacity of HUVECs were analyzed. For the cell migration test, 5×104 HUVECs in EBM-2 were cultured in the upper chamber (8 µm pore size, Milipore). We added the conditioned medium into the lower chamber. HUVECs cultured in EGM-2 served as control. After a 12-h incubation, migrated cells were fixed, stained, and counted. Capillary formation was assessed by culturing HUVECs in confocal plate coated with Matrigel (BD Bioscience) using conditioned medium or EGM-2. After 6 h incubation, the formed capillaries were stained with calcein AM (Invitrogen) and counted using a laser confocal scanning microscopy (Zeiss LSM710, Germany). 2.7. Animals and Disease Model of Hindlimb Ischemia. All animal studies were approved by the Animal Ethics Committee of Nankai University and followed the Tianjin Committee of Use and Care of Laboratory Animals. Eight–ten wks old male BALB/c nude mice (20–25 g) were purchased from the Laboratory Animal Center of the Academy of Military Medical Science (Beijing, China). A hindlimb ischemia model was established in BALB/c nude mice as previously described30. 2.8. Transplantation of ADSCs with Hydrogel. Animals used in this study were randomized into 7 groups as follows: sham, saline, CS-HA, CS-HA-IGF-1C, ADSCs, ADSCs/CS-HA, ADSCs/CS-HA-IGF-1C (n=10 for each group). Aggregation-induced emission (AIE) dots (1 nM) was used to pre-label ADSCs for cell tracking before delivery as previously described30. The labeled ADSCs were transplanted alone or with CS-HA scaffold, IGF-1C modified scaffold (1×106 ADSCs per 100 µl solution) by intramuscular injection at 3 sites into the ischemic hindlimbs of mice immediately after surgery. Mice receiving saline or CS-HA scaffold injection served as control. Most of mice appeared to be in good health during the study period. 7


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Mice were sacrificed at day 7 and 21 to harvest ischemic muscles. Then the muscle samples were cut into two parts from the middle, each of which was embedded in either optimal cutting temperature compound (Tissue-Tek) or paraffin. The tissues were cut into sections with a thickness of 6 µm. 2.9. Cell Fate Tracking by Fluorescence Imaging. To investigate in vivo survival and retention of transplanted ADSCs, we utilized the Maestro EX fluorescence imaging system (CRi, Inc.) to dynamically monitor the animals by placing the anesthetized mice on the equipped platform [exposure time = 200 ms, scans: day 1, 4, 7, 10, 14 and 21 postinjection, respectively]. Maestro software was used to remove the mouse background fluorescence. Besides, to observe cell survival in detail, frozen sections harvested at day 7 were rinsed thrice with PBS, counterstained with DAPI (Dapi-Fluoromount-G, Southern Biotech, England) and examined using a laser confocal scanning microscopy. 2.10. Blood Flow and Limb Salvage Measurements. To evaluate regenerative efficacy, animals were tracked by serial monitoring of hindlimb blood perfusion by Laser Doppler Perfusion Imaging System (Perimed AB, Sweden)30 at different time points (i.e. at day 0, 7, 14, and 21) post treatment. The blood flow from the knee joint to the toe region was quantified by analyzing digital color coded images, and perfusion rate was calculated. In addition, at day 21 post-injection, the percentages of three statuses including limb loss, foot necrosis, and limb salvage in animals have been also quantified by a blinded rater. 2.11. Histological Analysis. Hematoxylin and eosin (H & E) (at day 7 and day 21) and Masson’s trichrome (at day 21) staining were performed to evaluate infiltration of inflammatory cells and fibrosis of muscle tissue. The extent of inflammatory infiltration in 10 different random fields was calculated by a blinded rater. The area ratio of muscle degeneration and fibrosis in 10 different random fields was analyzed by a blinded rater. 2.12. Apoptosis Assay. Muscle cell apoptosis in the ischemic limbs was examined by TUNEL assay (Promega) according to a recent work24. 2.13. Immunofluorescence Staining. We performed immunofluorescence staining 8



on frozen section of muscle samples as reported previously24. The primary antibodies (purchased from Abcam) used in this study include: proliferating cell nuclear antigen (PCNA) (1:100), CD68 (1:100), CD31 (1:50), smooth muscle α-actin (α-SMA) (1:100), angiopoietin-1 (Ang-1) (1:100), collagen 1 (1:100), and basic fibroblast growth factor (bFGF) (1:200). Staining signals were visualized with the Alexa Fluor 633 or 488 conjugated secondary antibodies (1:200; Invitrogen). The sections were counterstained with DAPI (Southern Biotech, England) and examined using a laser confocal scanning microscopy. At least 10 different stained random fields were counted for each marker by a blinded rater. 2.14. Statistical Analysis. All data were presented as the means ± standard error of the mean (SEM). The statistical significance (P

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