Comparative Study of Heparin-Poloxamer Hydrogel Modified bFGF

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Comparative Study of Heparin-Poloxamer Hydrogel Modified bFGF and aFGF for In Vivo Wound Healing Efficiency Jiang Wu, Jingjing Zhu, Chaochao He, Zecong Xiao, Jingjing Ye, Yi Li, Anqi Chen, Hongyu Zhang, Xiaokun Li, Li Lin, Yingzheng Zhao, Jie Zheng, and Jian Xiao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b06047 • Publication Date (Web): 06 Jul 2016 Downloaded from http://pubs.acs.org on July 8, 2016

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ACS Applied Materials & Interfaces

Comparative Study of Heparin-Poloxamer Hydrogel Modified bFGF and aFGF for In Vivo Wound Healing Efficiency Jiang Wu#1, Jingjing Zhu#1, Chaochao He1, Zecong Xiao1, Jingjing Ye1, Yi Li1, Anqi Chen1, Hongyu Zhang1, Xiaokun Li, Li Lin1, Yingzheng Zhao1*, Jie Zheng2*, and Jian Xiao1* 1

School of Pharmaceutical Sciences Key Laboratory of Biotechnology and Pharmaceutical Engineering Wenzhou Medical University, Wenzhou, Zhejiang, 325035, China 2

Department of Chemical and Biomolecular Engineering The University of Akron, Akron, Ohio, 44325, USA

#

These authors contribute equally to this work.

*

Corresponding authors: Jian Xiao, [email protected]; Yingzheng Zhao, [email protected]; Jie Zheng, [email protected]

Keywords: Wound healing; Wound dressing; Fibroblast growth factor; Hydrogel; Heparin; Control release.

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Abstract Wound therapy remains a clinical challenge. Incorporation of growth factors (GFs) into heparin-functionalized polymer hydrogel is considered as a promising strategy to improve wound healing efficiency. However, different GFs incorporation into the same heparin-based hydrogels often lead to different wound healing effects, and the underlying GF-induced wound healing mechanisms still remain elusive. Herein, we developed a thermos-sensitive heparin-poloxamer (HP) hydrogel to load and deliver different GFs (aFGF and bFGF) for wound healing in vivo. The resulting GFs-based hydrogels with and without HP hydrogels were systematically evaluated and compared for their wound healing efficiency by extensive in vivo tests, including wound closure rate, granulation formation, re-epithelization, cell proliferation, collagen and angiogenesis expressions. While all GFs-based dressings with and without HP hydrogels exhibited the better wound healing efficacy than controls, both HP-aFGF and HP-bFGF hydrogels demonstrated their superior healing activity to improve wound closure, granulation formation, re-epithelization, and blood vessel density by up-regulation of PCNA proliferation and collagen synthesis, as compared to GF dressings alone. More importantly, HP-aFGF dressings exhibited the higher healing efficacy than HP-bFGF dressings, indicating that different a/bFGF surface properties lead to different binding and release behaviors in HP hydrogels, both of which will affect different wound healing efficiency. Based on experimental observations, the working mechanisms of different healing effects of HP-GFs on full skin removal wound were proposed. This work provides different views of the design and development of an effective hydrogel-based delivery system for GFs towards rapid wound healing.

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1. Introduction Wound healing therapy remains a great clinical challenge 1-3, because wound healing is a remarkably dynamic, complex, and interactive process, involving typical three overlapped phases of inflammatory phase, cell proliferation phase, and extracellular matrix remodeling phase 4-5. It is well-known that the angiogenesis growth factors (GFs) are critically important for regulating the wound repair process 67 , including cell migration and proliferation, extracellular matrix deposition, angiogenesis, and remodeling 8-9. Thus, significant efforts have been made to develop a wide variety of biocompatible materials (e.g. chitosan, collagen, alginate, peptide, and polyurethane) to incorporate GFs for maximizing their bioactivity in wound bed 10-12 . However, these common materials usually have several limitations, including poor anti-infective properties, low hydration environment, and frequent dressing exchanges 13-15, any of which would delay a whole wound healing process. Moreover, the fundamental understanding of the topical application of growth factors to accelerate wound healing still remain largely unknown 16. Hydrogels, containing 80-90% water and 3D porous structure, are considered as promising dressing materials to load and deliver GFs for wound treatments 17-20. In general, different hydrogels have been developed to contain some functional groups for specifically interacting with the loaded molecules to realize its control-release behaviors in vitro and in vivo 21-26. Such surface modifications facilitate binding specificity and affinity between modified groups with GFs, and help to remedy the biochemical properties of the conjugated biomolecules, such as water-solubility, bioavailability, selectivity, and even toxicity 27-29. Among these, heparin is often used as a bridge to modify GFs to achieve their combinatorial activity 30-32. Briefly, heparin is a negative linear polysaccharide 33 that can easily bind to GFs domains (namely heparin-binding GFs 34) containing rich positively charged lysine and arginine residues 35-36. Such heparin-GFs binding helps to not only stabilize GFs 37, but also enhance GFs binding affinity to cell-surface receptors to initiate more intracellular signaling pathways 38. Hence, use of heparin-based hydrogels to incorporate different growth factors and cytokines through their heparin-binding domain is the most possible efficient way to form a (multi)functional complex 37, 39. Several heparin-GF hydrogel systems have been reported as proof-of-concept models for wound healings, including a star PEG-heparin 3D hydrogel with both bFGF and VEGF 40-41, chitosan/pluronic hydrogel containing heparin/bFGF 42-43, heparin-alginate bFGF hydrogels 44, and NGF loaded thermosensitive heparin-poloxamer (HP) hydrogel 45-46. Since all these heparin-GF hydrogels exhibit different degrees of wound healing improvement in vivo or in vitro, it appears to be a common opinion that regardless of different GFs, the same heparin-based materials can always interact with GFs and load GFs into heparin host materials due to the presence of heparin binding domain 4749 . However, in our opinion, expect the heparin binding domain of the GFs, different GFs of different structural properties (surface charge intensity changes, hydrophobicity and hydrophilicity 50) would also affect the binding and releasing behaviors of GFs in host materials. There is little attention to the structural-dependent effect of different GFs on their binding/release behaviors in host materials, as well as their consequences on wound healing. For instance, aFGF and bFGF are the two most commonly used heparin binding GFs, both of which show similar chemical structures, but a major difference in isoelectric point (IP). aFGF has 140 amino acids, 15.8 KDa 3



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in molecular mass, and 5.6 of IP, showing acidity in saline 51, while bFGF has 146 amino acids, 16.4 KDa in molecular mass, 9.6 of IP, showing alkaline in saline 52-53. This totally different acid-base property between aFGF and bFGF will lead to different surface charges 54, especially when loading into heparin-based hydrogels in physiological saline solution. It is reasonable to speculate that aFGF and bFGF will have different interactions with heparin-based materials in physiological saline solution, probably leading to different GF-induced wound healing effects via different binding and release behaviors between GFs and host heparin-based materials. To examine this hypothesis, here we synthesized heparin-poloxamer (HP) hydrogels, followed by modification of HP hydrogels with different aFGF and bFGF to better understand different GFs/hydrogel-induced wound healing effects in vivo. Particularly we focused on the investigation and comparison of different GFs/hydrogel-induced wound healing effects between free aFGF and bFGF, between HP-aFGF and HP-bFGF, and between a/bFGFs and the corresponding HP-a/bGFs. In vivo wound efficacy was assessed by C57BL/6 mice using full-thickness wound. While all FGF-based dressings stimulated and improved wound healing in vivo, wound healing efficiency was largely different. Specifically, both HP-GF hydrogels exhibited superior wound healing activity as compared to free a/bFGF in terms of wound closure, granulation formation, re-epithelization, and blood vessel density. Moreover, HP-aFGF outperformed HP-bFGF to achieve the better wound-healing capacity because aFGF can be efficiently released from HP hydrogels. The results suggest that this rationally designed construct is critical for better understanding biomaterial mediated wound healing and rational design of wound healing biomaterials. 2. Materials and Methods 2.1. Preparation of HP-aFGF hydrogels and HP-bFGF hydrogels Heparin–poloxamer conjugate was prepared according to EDC/NHS method reported in previous paper 55. HP-aFGF hydrogels and HP-bFGF hydrogels containing different amounts of HP and aFGF / bFGF were prepared using the cold method 56. In brief, lyophilized HP powder was mixed with aFGF / bFGF (the Key Laboratory of Biotechnology and Pharmaceutical Engineering, Wenzhou Medical University, China) at 4 oC under gentle stirring. The mixture was kept in a refrigerator at 4 oC overnight, and finally to obtain a clear solution. 2.2. Micromorphology of hydrogel system The micromorphology of the dehydrated HP, HP-bFGF, HP-aFGF hydrogels was observed on a scanning electron microscope (SEM; Hitachi, H-7500, Japan). The hydrogels were freeze and lyophilized by vacuum freeze dryer for 24 h. The dehydrated specimens were cross-sectioned and sputtered with gold, and their surface of solid was observed by scanning electron microscope. 2.3. Rheological behavior of HP and HP-GFs hydrogels Rheological measurements of the HP, HP-aFGF, and HP-bFGF were carried out using the rheometer (TA-AR-G2) at different temperatures from 10 to 40 oC. The elastic modulus and viscous shear modulus were measured using the flat plates (12 mm), and shear frequency was set to 10 rad/s, shear strain was set to 1%. 4


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2.4. Releasing profile of aFGF / bFGF from HP-aFGF / HP-bFGF HP-GFs were prepared as before. At each time point (0h, 6h, 12h, 24h, 3 day, 8day, 14day and 17 day), GFs were collecting through the supernatant, then replaced by fresh solution. The released GFs was analyzed by specific GF enzyme-linked immunosorbent assay Kit (ELISA, Westang system, Shanghai, China). The amount of released GFs was normalized through the former and final concentrations. The GFs release system was carried out at 37 oC. This trend of the release profile was maintained up to the end of the experiment. 2.5. In vivo wound healing model 6-7 weeks male C57BL/6 mice were purchased from the Animal Center of Chinese Academy of Sciences, Shanghai, China. The experiment animals were carried out with the National Institutes of Health Guide Concerning the Care and Use of Laboratory Animals. All animal experiments were performed in accordance with the guidelines approved by the Animal Experimentation Ethics Committee of Wenzhou Medical University, Wenzhou, China. Mice were maintained for at least 7d before the experiment on a standard diet and water was provided freely available. Temperature (23 oC -25 oC), humidity (35−60%), and photoperiod (12 h light/dark cycle) were kept constant. All the animals were anaesthetized by an intraperitoneal injection of 4% chloral hydrate (0.1 ml/10g) and the dorsal area was shaved. 0.5-mm-thick silicone donutshaped splints (the external diameter of 16 mm, the internal diameter of 8 mm) were fixed on either side of the dorsal midline using 6-0 Prolene suture. Two full-thickness cutaneous wounds were made using a 6 mm round skin biopsy punch (Acuderm® inc., Ft Lauderdale, FL, USA). The forty-two C57BL/6 mice were randomly selected for each group. Saline, vehicle alone, 1.6µg free aFGF/ bFGF or HP-aFGF/ bFGF hydrogels was administered to wounds (n = 7 animals/14 wounds per group) as a 10 µl solution. To avoid cross-contamination, all wounds received the same treatment. The wounds of the treated animals were applied a Tegaderm™ transparent dressing (3M Health Care, Germany) and wrapped with self-adhesive wrap to deter chewing of the splints. All the animals were fed in individual cages with food and water ad libitum, and watched every day during the total period of the experiment. At days 0, 7, 10, 14, and 17 post-treatments, the wound closure rate was determined by measuring the wound area by Image-Pro plus to trace the wound margin. (Wound closure rate (%) = (wound areaday0 - wound areaday#)×100%/wound areaday0). At day 3 or 17 postsurgery C57BL/6 mice were sacrificed, and wounds with surrounding tissue were harvested for histological evaluation. 2.6. Histological analysis For histological preparation the skin was fixed in 4% paraformaldehyde in 0.01M phosphate buffered saline (PBS, PH = 7.4) overnight and paraffin embedded. Skin tissue was sectioned into 5-µm thickness slices for Hematoxylin and Eosin (H&E) (Beyotime Institute of Biotechnology, China) staining and for collagen formation by Masson’s trichrome staining (Beyotime), using standard procedures. Sections were analyzed and images were captured using a Nikon ECLPSE 80i (Nikon, Japan). 2.7. Immunohistochemical staining Wound sections were fixed with 4% paraformaldehyde solution for 24 h and dehydrated in graded ethanol series, then embedded in paraffin. Skin tissue sections, 5



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prepared by a microtome to 5-µm thickness, were deparaffinized and rehydrated, and then immersed in 3% H2O2 and 80% carbinol for 15 min at room temperature to block the endogenous peroxidase activity. The tissue sections were heated to antigen recovery in 10 mM sodium citrate buffer (pH 6.0) and after washing the samples were blocked using 5% bovine serum albumin (BSA) (Beyotime) for 30 min at room temperature. Primary antibody rabbit polyclonal anti-cytokeratin (ab9377, 1:75, Abcam), mouse monoclonal anti-PCNA (sc25280, 1:200, Santa Cruz Biotech, CA, USA), rabbit polyclonal anti-collagen III (ab7778, 1:300, Abcam), rabbit polyclonal anti-collagen I (ab21286, 1:300, Abcam) and tagged secondary antibody goat antimouse or goat anti-rabbit, were used. The tissue was treated with DAB chromogen kit (ZSGB-BIO, Beijing, China), then counterstained with hematoxylin (Beyotime Institute of Biotechnology, China) for 5 min. Day 17 skin sections were stained with rabbit polyclonal anti-CD31 (ab28364, 1:200, Abcam) followed by goat anti-rabbit IgG Alexa Fluor® 647-conjugated secondary antibody (ab150083, 1:1500, Abcam) and nuclei with DAPI (Beyotime). All fluorescent images were taken using a Nikon confocal laser microscope (Nikon, A1 PLUS, Tokyo, Japan). 2.8. Statistical analysis All statistical data are expressed as means ± standard deviation (SD). Statistical analysis of all data was performed one-way analysis of variance (ANOVA) followed by Tukey’s test with GraphPad Prism 5 software (GraphPad Software Inc., La Jolla, CA, USA). For all comparisons, p values < 0.05 were considered to be statistically significant. 3. Results and Discussion Considering that the wound-healing process generally involves three overlapping stages: the inflammatory stage, the proliferative stage, and the remodeling phase, here we systematically tested our hydrogel dressings on wound closure, granulation formation with collagen deposition, re-epithelialization, and neovascularization as well as protein related cell proliferation, collagen I and collagen III synthesis upon wound. 3.1. Characterization of HP-aFGF and HP-bFGF hydrogels Since copolymer heparin-poloxamer (HP) exhibited a thermal-sensitive sol-gel phase transition achieved by poloxamer’s PEO-PEG ratio, HP was used as smart biomaterials to load and delivery both aFGF and bFGF for wound regeneration tests. As shown in Fig. 1A, HP formed hydrogels upon heating to a body temperature of 37 °C, while melting to sols when cooling down to 4 °C. This sol-gel transition first allows GFs to well mix in HP solution at 4 °C. Then, when applying the GF-HP solution to in vivo wound bed at 37 °C, HP gelation occurs, making GFs to be successfully loaded into HP hydrogels (namely as hydrogels thereafter). Rheology tests were conducted to reveal the gelation process of HP-based hydrogels by varying temperatures (Fig. 1B). When a hydrogel exhibits viscoelastic behaviors, the storage (G′) and loss (G'') moduli are used to measure its elastic and viscous components. Figure 1B showed that all HP-based hydrogels, regardless of loading of GFs, displayed a similar sol-to-gel transition at around 27 oC. When temperatures was above 27 oC, both G′ and G″ values of the HP-based hydrogels increased rapidly as temperatures. The transition temperature of 27 oC is close to room temperature as well, making HP-based hydrogels be suitable for biomedical applications. Similar 6


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rheologcial results among different HP-based hydrogels also indicate that loading of GFs into HPs does not significantly change the rheological properties of HP-based hydrogels. Fig. 1C shows the micromorphology of HP, HP-aFGF, and HP-bFGF hydrogels by SEM. SEM images did not show any obvious differences in porous structures of HP hydrogels in the absence and presence of loaded GFs. This because within the length scale of hundred micrometers in SEM images, a very small loading of GFs (~1.6 µg) into HP hydrogels (~1800 µg) will not cause significant changes in porous structures of HP hydrogels. Moreover, inside all HP-based hydrogels, there existed many sponge-like structures interconnected with each other, which allow growth factors to diffuse through these 3D network structures and realize release. It should be noted that all HP-based dressings can attach easily to the wounds with no need for biological adhesives, due to the gelation structure and high water content (>80%) of HP hydrogels, thus allowing the wound to stay desirably hydrated. In addition, we also tested the biocompatibility of HP hydrogels using 3T3 fibroblast cell lines that is the most commonly used cell line for wound treatment. Light microscope images (Fig. S1A, Supporting Information) showed that at 0.1 wt% and 1 wt% HP concentration, fibroblast cells were able to retain their healthy morphologies similar to those in the control group. Consistently, Figure S1B quantitatively showed that HP hydrogels of 0.1 wt% and 1 wt% were not toxic to cells, with >100% cell viability, indicating the excellent biocompatibility and safety of HP hydrogels in vitro. 3.2. HP-GFs accelerate wound closure in mice To evaluate the effect of HP-GFs in skin wound regeneration process, aFGF, bFGF, HP-aFGF, and HP-bFGF dressing were applied to the full-thickness cutaneous C57BL/6 mice model, in comparison to saline and HP dressings as controls. Fig. 2A shows the side-by-side comparison of different GF-treated wounds on day 7, 10, 14, and 17, respectively. Visual inspection of the wound closure in mice revealed that unlike the negative control that exhibited almost no reduction in wound size during the whole treatment period (17 days), all of other tested dressings displayed the noticeable reductions in the wound sizes on days 14 and 17. Particularly, the wound closure was greatly enhanced with HP-bFGF and HP-aFGF dressings. Quantitatively, Fig. 2B presents the wound closure rate for each case, as sampled from at least six replicates of the wounds. Consistent with visual inspection of wound closure in Fig. 2A, HP-aFGF showed the highest wound closure rate at every time point and achieved a final wound closure rate of 96.1±6.4% on day 17, as compared to the wound closure rate of the other dressings in a decrease order: HP-bFGF (91.3±10.9%), bFGF (72.9±11.8%), aFGF (68.8±9.3%), HP (65.8±13.6), and control (60.8±18.4). More importantly, upon addition of aFGF or bFGF to HP, the healing efficiencies of both HP-aFGF and HP-bFGF were increased as compared to those of pure aFGF and bFGF in the absence of HP, and such HP-enhanced wound healing effect became more pronounced in the case of HP-aFGF (Fig. 2C). It was also reported that aFGF alone is 10-100 fold less potent than bFGF in inducing wound related cell proliferation 57. These data indicate that synergetic interactions between HP and aFGF offer the more efficient suturing effect at the wound site, thus promoting wound closure during the early stage of the wound-healing process. 3.3. HP-GFs improve granulation formation Granulation tissue formation generally starts to appear at the wound site in 2-5 days after injury 58. Fig. 3 shows the H&E staining of wound histology sections 7



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treated with HP, HP-aFGF, and HP-bFGF dressings. At day 7, no obvious neoepidermis was observed underneath the eschar of all groups. While the length of wound closure for all groups showed no significant difference, the thickness of the granulation was significantly different between all groups. Both control and HP groups had the thinnest granulation layers (Fig. 3A-B). HP-GFs (Fig. 3D and F) had far thinker and more continuous layers of granulation across the entire wound gap than GFs alone (Fig. 3C and E), indicating that the incorporation of GFs into HP hydrogels improves the granulation formation. At day 17, neoepidermis formation were observed in all groups, though both HP and control groups still retained the larger un-closure areas at the wound site. The thickness of the neoepidermis followed the same orders (from the thickest to the thinnest): HP-GFs groups (Fig. 3J, L) >GFs groups (Fig. 3I, K)>control groups (Fig. 3G, H). Consistently, HP-aFGF group showed a thicker epidermis than HP-bFGF group. 3.4. HP-GFs enhance collagen deposition Collagen deposition is a critical factor to determine the strength and appearance of the scar that has formed 59-61. Fig. 4 shows collagen deposition in the dermis of regenerated skin for all six groups at day 7 and 17 using Masson Trichrome Staining (MTS). At day 7, all six groups showed different amounts of collagen deposition, among which HP-GFs-treated wounds exhibited the highest collagen deposition (as indicated by the strongest the blue staining intensity of regenerated collagens, Fig. 4D, F), while two control groups had the least collagen deposition (Fig. 4A, B). At day 17, difference in collagen deposition became even more pronounced. HP-aFGF-treated wounds had the most densely packed collagen fibers running in parallel arrangement in all groups (Fig. 4L), while HP-bFGF-treated wounds took the second place in collagen deposition with similar parallel arrangement (Fig. 4J). Compared to HP-GFs groups, the other four groups appeared to have loosely packed collagen fibers running in irregular arrangements (Fig. 4G, H, I, K). Consistently, among six groups, the HPGF dressings have promoted mature granulation tissue formation with dense collagen deposition, where HP-aFGF shows the relatively higher uniform collagen alignment than HP-bFGF. 3.5. HP-GFs activate cytokeratin expression related to re-epithelialization Furthermore, immunohistochemical staining of re-epithelialization related cytokeratin were taken to evaluate the re-epithelialization of wound regeneration. HPaFGF (Fig. 5F, L) and HP-bFGF (Fig. 5D, J) groups exhibited the faster reepithelization than GFs (Fig. 5C, E, I, K) and control groups (Fig. 5A, G) on 7 and 17 days, as evidenced by the early expression of more cytokeratin in the thicker epidermis. While both HP-GFs groups promoted the formation of skin appendages from day 7 to day 17, HP-aFGF-treated wounds exhibited the much higher number of skin appendages and the thicker epithelial layers than HP-bFGF-treated wounds (Fig. 5J and L). Additionally, a close-up visualization also revealed the regeneration of hair follicles in the wounds treated with HP-GFs groups, demonstrating the fast healing effect of HP-GFs on the re-epithelization of wounds in vivo. On the other hand, GFs groups actually did not show large differences from control groups regarding the formation of epithelization and epidermis. Thus, the epithelization of wound incisions increased in the order of HP-aFGF and HP-bFGF>GFs>control. Taken together, both sustaining release of aFGF and bFGF are known to promote wound healing. When incorporating GFs into HP hydrogels, HP hydrogels not only 8


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retain and prolong the bioactivities of GFs, but also offer a hydrated environment to greatly accelerate wound healing process, as demonstrated by fast wound closure, earlier formation of granulation tissue, epithelization, thicker epidermis, and dense and uniform collagen deposition. 3.6. HP-GFs induce cell proliferative activities and collagen synthesis In order to better understand wound healing mechanisms, here we mainly used immunohistochemistry of tissue sections to study the proliferative activities of cell by detecting the expression level of cell nuclear antigen (PCNA) as a biomarker. It can be seen that HP-GFs groups had up-regulated the more PCNA positive keratinocytes at the wound edge than other groups at day 7, indicating a higher cellular proliferation of HP-GFs (Fig. 6A, HP-aFGF, p bFGF (565.7±109.8 for collagen I, 73.3±6.7 for collagen III) > aFGF (550.2±56.6 for collagen I, 66.6±8.5 for collagen III) > HP (251.2±119.6 for collagen I, 21.3±5.2 for collagen III) and control (252.8±67.5 for collagen I, 18.9±3.4 for collagen III). The enhanced granulation formation as shown in H&E and MTS were mainly attributed to the collagen synthesis at the early stage of wound regeneration process. These results reveal that the sustained release of GFs from HP hydrogels plays a continuous role in stimulate the protein expression of PCNA and collagen I/III at wound sites, which in turn promotes the formation of new provisional ECM for cell proliferation and cell migration. 3.7. HP-GFs boost angiogenesis Proper wound healing requires angiogenesis of newly-generated dermis. To evaluate neovascularization of the wounds, the newly-formed vessels at both wound bed and edge were characterized by CD31 (cluster of differentiation 31) staining, a vascular endothelial cells marker on day 17 64, from which the average vessel density is quantified in Fig. 8. Visual inspection of immunofluorescence of CD31 and DAPI on wound bed and wound edge (Fig. 8A and Fig. 8B) confirmed the large amount of newly formed vessels in HP-GFs groups (Fig. 8D, F, J and L), but only a few or no CD-31 expression of vascular cells was found in control (Fig. 8A and L), HP (Fig. 8B and H), and GFs groups, suggesting that HP-GFs boost angiogenesis and lead to a fast healing. Quantitatively, the average vessel density was quantified in Fig. 8C, the vessel density of the HP-aFGF group was found to be the highest (36.7±2.5 per field in wound edge, 34.3±5.9 per filed in wound bed), higher than that of the HP-bFGF 9



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group (26.3±3.3 per field in wound edge, 24.7±1.2 per field in wound bed), bFGF group (24.1±1 per field in wound edge, 18.7 ±4.7 per field in wound bed), and aFGF group (22.3±3.5 per field in wound edge, 16.7±2.2 per field in wound bed). Moreover, the vessel densities of both HP-GFs groups were always significantly higher than those other groups. Both control and HP groups had almost undetectable CD-31 at day 17. These results further confirm that incorporation of GFs into HP hydrogels has large positive effect on vessel generation. Additionally, HP-aFGF group were also found to have more active CD-31 expression than HP-bFGF, demonstrating a better effect of HP-aFGF on neovascularization than HP-bFGF. 3.8. Mechanistic model of HP modifying aFGF versus bFGF Both aFGF and bFGF are well known important class of growth factors, which act as a crucial extrinsic cue for stem cell maintenance and differentiation, possesses angiogenesis, and neuroprotective properties. However, aFGF and bFGF also suffer from their low stability caused by enzymatic degradation that greatly limits their use in clinical applications. Since HP contains specific binding sites for FGF, which stabilizes this protein within the ECM, we developed the HP hydrogel to load and stabilize aFGF and bFGF and performed a series comparative study of their wound healing efficiency in vivo. Collective data have showed that GF-loaded HP hydrogels are extremely more effective than free GFs, due to the protection of HP hydrogel matrix. More importantly, while both GF-loaded HP hydrogels can achieve fast wound closure, granulation tissue formation, collagen deposition, and reepithelialization due to the higher PCNA expression with specific collagen synthesis and angiogenesis, it is interesting to observe an opposite trend in overall wound healing efficiency between free aFGF and bFGF hydrogel and between HP-aFGF and HP-bFGF hydrogels. In the absence of HP hydrogels, free aFGF has the lower wound healing efficiency than free bFGF, but when incorporation of a/bFGF into HP hydrogels, HP-aFGF showed the higher wound healing efficiency than HP-bFGF. Such sharp difference has not been studied before, so based on our findings we proposed a mechanistic model to interpret the wound healing differences between free FGFs and HP-loaded FGFs (Figure 9). Free aFGF and bFGF have very similar structures, but different surface charge in physiological saline solution due to different isoelectric point (IPaFGF = 5.6 and IPbFGF = 9.6) 54, resulting in aFGF being negatively charged and bFGF being positively charged on surface. HP itself is negatively charged due to the presence of heparin. So, it is clear that the loading of a/bFGF into HP hydrogels would change the interactions between a/bFGF and HP and the release rate of a/bFGF from HP. As illustrated in Fig. 9A, during the formation of HP-GF hydrogels in stage I, bFGF can bind stronger and easier to HP than aFGF due to favorable electrostatic interactions. But, such strong binding between bFGF and HP, in turn, causes the difficulty to release bFGF from HP during the release stage II. Fig. 9B clearly shows substantial difference in the release profiles from the two HP-GFs systems. While both HP-GFs hydrogels demonstrate a sustained release of a/bFGF for over 10 days, more aFGF (42.8%) is released HP-aFGF than bFGF (24.3%) released from HP-bFGF, confirming that binding of different FGFs to heparin modulates their release from the heparin-containing hydrogels. To better understand the release behavior of GFs from HP hydrogels, we designed two different experiments to examine the degradation of HP hydrogels by either immersing whole hydrogels into bulk water or making the 10


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bottom face of the hydrogel in contact with water. Within 10 days, no obvious degradation was observed in both cases, but slight gel dissolution occurred at the hydrogel-water interface (Figure S2 in Supporting Information). We should mention that the degradation of HP hydrogels is unlikely to occur at the wound interface, simply because the water content at the hydrogel-wound interface would be much less than that in bulk water we tested. Thus, the release of GFs from HP hydrogels is mainly caused by a combined release behavior of GF diffusion and gel dissolution, but not by HP degradation. Different a/bFGF surface properties in saline solution lead to different binding and release behaviors in HP hydrogels, which are attributed to different wound healing efficiency. 4. Conclusions In this work, HP-GF hydrogels were designed and synthesized to demonstrate their improved wound healing capacity in vivo. Upon incorporation of aFGF and bFGF into HP hydrogels, in vivo wound healing results showed that Upon incorporation of aFGF and bFGF into HP hydrogels, both HP-GF hydrogels accelerated the wound closure of full-thickness excisional wounds of mice with enhanced granulation tissue formation during initial stages of healing and stimulated angiogenesis and re-epithelization through the proliferation stage. Furthermore, both HP-GF hydrogels prompted expression of cell proliferation and collagen synthesis, all of which are beneficial for vascularization. Moreover, due to the different surface charge properties between aFGF and bFGF, positively charged bFGF had the stronger interactions with negatively charged HP than negatively charged aFGF, which makes bFGF difficult to be released from HP hydrogels leading to the less wound healing efficiency. Such different interaction modes between aFGF-HP and bFGF-HP hydrogels interpret not only the FGF binding and release behaviors, but also FGFinduced wound healing effect. Irrespective of the mechanistic details, enhancement of wound healing by HP-GF-based materials is likely to be the combination of (i) a GF that can recognize and interact with specific GF receptors to stimulate wound healing, (ii) a biocompatible HP platform that provides solid supports to stabilize loaded proteins and facilitate GF binding, and (iii) a synergetic healing effect of GF-HP materials. While many studies have reported that FGFs stimulate angiogenesis and wound healing, this work reveals the possible structural-dependent interactions between different FGF and HP on wound healing effect, and hopefully this information will be carefully considered for future development of heparin-based polymers with GFs for wound healing applications. In addition, other structural properties should also be considered for the future design of GFs for wound healing, including ionic property, surface charges, surface hydrophobicity, surface modification ratio, and cytokine activate/binding sites with GFs receptors upon modification. Hopefully, this work will lead to the structural-based design for new GF-based materials for wound healing. Acknowledgement. J.W. is grateful for the fundings from Zhejiang Provincial Natural Science Foundation of China (LQ15E030003), Wenzhou Science & Technology Bureau of China (Y20140727), and the Opening Project of Zhejiang Provincial Top Key Discipline of Pharmaceutical Sciences (YKFJ001). J.X. thanks for the fundings from Zhejiang Provincial Program for the Cultivation of High-level Innovative Health talents, National Natural Science Funding of China (81372112, 81372064, 81302775, 81472165, 81571923), Zhejiang Provincial Natural Science 11



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Foundation of China (Y14H150023, Y14H090062, LY13H030008, LY14H150008), State Key Basic Research Development Program (2012CB518105), and Zhejiang Provincial Project of Key Group (2010R50042). J.Z. thanks financial support, in part, from the National Science Foundation (CAREER Award CBET-0952624, CBET1510099, DMR-1607475), Alzheimer Association New Investigator Research Grant (2015-NIRG-341372), and National Natural Science Foundation of China (NSFC21528601).

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Figure 1. (A) Temperature-sensitive characteristics of HP hydrogels at 4 oC, 37 oC, and 4 oC. (B) Storage (G′) and loss (G'') moduli of different HP-based gels as a function of temperature from 40 to 10 oC. (C) SEM images of HP (c1), HP-aFGF (c2), and HP-bFGF (c3). Scale bars in c1-c3 are 200 µm.

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Figure 2. HP-GFs accelerate wound closure. (A) Wound closure in C57BL/6 mice treated with normal saline, HP, bFGF, HP-bFGF, aFGF, and HP-aFGF at day 0, 7, 10, 14, and 17. Unit is mm. (B) Wound closure rates of control, free aFGF and bFGF, and HP-aFGF, HP-bFGF groups. (C) HP-induced wound closure increment as calculated by the difference between the area under curve of HP-aFGF and aFGF versus HPbFGF and bFGF. Bars indicate means ± SD, *** P < 0.001.

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Figure 3. Hematoxylin and Eosin (H&E) images for different groups in wound healing at day 7 (A, B, C, D, E, F) and day 17 (G, H, I, J, K, L). Scale bars = 500 µm.

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Figure 4. Histochemical staining of collagens during post-wounding at day 7 (A, B, C, D, E, F) and day 17 (G, H, I, J, K, L). Tissue sections are stained with Masson’s trichrome (blue = collagen; red = cytoplasm and muscle fibers). The nucleus was counterstained with hematoxylin (violet). Rectangles refer to the close-up areas. Original scale bar = 500 µm and close-up scale bar = 100 µm.

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Figure 5. Representative light microscopy images of wound center sections stained with immunohistochemically for cytokeratin (IHC-Cytokeratin, a marker of epithelial cells) at day 7 (A, B, C, D, E, F) and day 17 (G, H, I, J, K, L). Arrows indicate newly formed dermal appendages and hair follicles within granulation tissues. Bar = 500 µm.

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Figure 6. (A) Representative images of PCNA immunohistochemistry staining on day 7 of post-wounding in mice, showing enhanced cellular proliferation as evidenced by increased expression of PCNA. Red dotted lines are used to separate the epidermis and the dermis. Scale bar = 50 µm. (B) Quantitative analysis of relative density of PCNA at 7 d after surgery, *** P < 0.001, ** P < 0.01, * P < 0.05, n = 3.

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Figure 7. Immunohistochemistry staining images for (A) collagen I and (C) collagen III on day 7 of post-wounding in mice. Red dotted lines are used to separate the epidermis and the dermis. Scale bar = 100 µm, magnification scale bar = 50 µm. Quantitative analysis of relative density of (B) collagen I and (D) collagen III at 7 d after surgery. *** P < 0.001, ** P < 0.01, * P < 0.05, n = 3.

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Figure 8. New blood vessels stained with CD31 (red) and DAPI (blue) in (A) wound edge and (B) wound bed at day 17 postoperative. Scale bar = 100 µm, magnification scale bar = 50 µm. Quantitative analysis of number of vessels pre field at 17 day after surgery in (C) wound edge and (D) wound bed. *** P < 0.001, ** P < 0.01, * P < 0.05, n = 3.

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Figure 9. (A) Scheme of different interactions between HP-aFGF and HP-bFGF. Positively charged bFGF binds stronger to negatively charged HP than negatively charged aFGF due to favorable electrostatic interactions caused by different PIs of aFGF and bFGF. (B) Release profiles of aFGF from HP-aFGF hydrogels and bFGF from HP-bFGF hydrogels.

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Comparative Study of Heparin-Poloxamer Hydrogel Modified bFGF

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