Self-Assembling Peptide Nanofibrous Hydrogel on Immediate

Sep 8, 2015 - Based on the definition for the critical size of a bony defect,(26) round-shaped ilium defects were produced as follows: two holes (4 mm...
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Self-Assembling Peptide Nanofibrous Hydrogel on Immediate Hemostasis and Accelerative Osteosis Min Wu,*,†,‡ Zhaoyang Ye,‡ Hongyan Zhu,§ and Xiaojun Zhao*,‡,∥ †

Huaxi MR Research Center (HMRRC), Department of Radiology, West China Hospital of Sichuan University, Chengdu, 610041 Sichuan, China ‡ Institute for Nanobiomedical Technology and Membrane Biology, West China Hospital of Sichuan University, Chengdu, 610041 Sichuan, China § Laboratory of Stem Cell Biology, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, 610041 Sichuan, China ∥ Center for Biomedical Engineering, NE47-379, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139-4307, United States ABSTRACT: The use of local agents to achieve hemostasis of bone that does not interfere with repair and recovery is a complex and emergency subject in surgery. In this study, the dual functional biodegradable self-assembling nanopeptide (SAP) RADA16-I was synthesized by solid phase synthesis and was shown to exhibit immediate hemostasis and accelerative osteosis. The RADA16-I showed good performance as a hemostatic agent, which was investigated by comparison with the effects of bone wax in the ilium bone defect model of New Zealand rabbits. The RADA16-I exhibited efficient function of bone regeneration in both radiographic analysis and histological examination, while the bone wax inhibited osteogenesis. Moreover, in in vivo experiment, the RADA16-I was shown to exhibit excellent biocompatibility, while the group with bone wax showed a severe inflammatory response at the interface with bone. Thus, the RADA16-I is proven to be an excellent biocompatible material with effective dual function of hemostasis and osteosis.

1. INTRODUCTION Bone wax has been widely used for controlling bleeding from bony structures during most surgical procedures since its formal introduction in surgery by Horsely in 1892.1 This is mainly because it is simple, hemostatically efficacious, inexpensive, and easy to use. However, increasing evidence has shown that bone wax may cause foreign body reactions, infections, and osteogenesis inhibition.2,3 Additionally, the occurrences of quadriplegia after spine surgery,4 lower extremity paralysis after thoracotomy,5 sigmoid sinus obstruction after mastoid surgery,6 and cerebrospinal fluid leakage after cranial base surgery7 have mainly been caused by the use of bone wax. Controlling bleeding from bony structures is still a great challenge, because it is difficult to find bleeding sites and the injured bony tissue is rigid and irregular. An ideal bone hemostatic agent would rapidly induce hemostasis and improve bone-healing, while it would be fully resorbable, noninflammatory, biocompatible, and inexpensive, as well as easy to use.8 Several alternative hemostatic agents, including oxidized regenerated cellulose, gel sponges, and collagen fleece, have been used in clinical practice.9 However, these hemostatics often are often inconvenient to use and block bone-healing in the surgical sites;3 additionally, these local hemostatic agents © XXXX American Chemical Society

are not fully free from risk of complications, which can be attributed to mechanical compression or pathological effects. RADA16-I is an ionic and self-complementary nanopeptide (SANP) that consists of alternating hydrophilic and hydrophobic amino acids residues. Under physiological conditions, this peptide can spontaneously assemble into well-ordered nanofibers and hydrogels that contain over 99% water in situ.10,11 The three-dimensional nanofiber structures of the peptide hydrogel are similar to the naturally occurring extracellular matrix (ECM) that supports cell and tissue growth and consequently repairs tissue injury.12−14 This peptide hydrogel has also been reported to support rat hippocampal cell attachment and proliferation15−20 and promote the differentiation of adult progenitor cells into functional hepatocyte-like spheroid clusters.21 In addition, Ellis-Behnke et al. reported that the SANP can establish a nanofiber barrier to immediately achieve complete hemostasis when applied directly to a parenchyma wound in the brain, spinal cord, femoral artery, and liver.22 Misawa et al. Received: April 14, 2015 Revised: August 25, 2015

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DOI: 10.1021/acs.biomac.5b00493 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules reported that this self-assembling peptide also facilitates bone regeneration for supporting cells during penetration and engrafting into calvaria bone defects in mice.23 These combined features make RADA16-I a potential ideal material for both rapid hemostasis and improved bone healing.24 Herein, we describe the design of RADA16-I and evaluate its effect on hemostasis and osteosis in the ilium bone defect model of the New Zealand rabbit.

sacrificed from an overdose of sodium pentobarbital at 4, 8, and 12 weeks. 2.4. New Zealand Rabbit Ilium Defect Model. A 4 mm diameter, round-shaped defect was made in the ilium of a New Zealand Rabbit to serve as a bony structure hemostatic model during this study.25 A 3 cm incision was made in the skin of the anterior superior iliac spine of a 6 month old adult rabbit; the muscular dissection proceeded down to the periosteum of the ilium, and then the periosteum was carefully peeled off using a periosteal raspatory (Figure 1A).

2. MATERIALS AND METHODS 2.1. Materials. The RADA16-I peptide (AcN-RADARADARADARADA-CNH2, theoretical mass = 1713 g/mol, 99.5% purified powder) was synthesized commercially using solid phase synthesis methods by the Shanghai Bootech BioScience & Technology Co., Ltd., Shanghai, China. Peptide stock solutions were prepared by dissolving the peptide powders in Milli-Q water (18.2 MΩ) to a concentration of 10 mg/mL, mixing, sonicating for 30 s, filtering, and then storing at 4 °C. For the AFM measurement, the peptide stock solution was diluted to a concentration of 0.17 mg/mL (100 μM). The bone wax (Ethicon Johnson & Johnson, Somerville, NJ) remained sealed until implantation. 2.2. Atomic Force Microscopy. A 1 μL aliquot of the peptide solution (100 μM) was deposited evenly onto a freshly cleaved mica sheet for approximately 30 s; then, the sheet was washed twice using 100 μL of Milli-Q water to remove the unattached peptides. After air-drying, AFM images were collected at room temperature using an AFM (SPI4000 Probe Station and SPA-400 SPM Unit, Seiko Instruments Inc., Chiba, Japan) operating in the dynamic force mode. A 20 μm scanner, Si-DF20 microcantilever, and Si tip (radius = 10 nm) were employed during the AFM observation; the typical scan parameters were set as follows: amplitude 1 V, integral gain 0.10−0.45 V, proportional gain 0.03−0.15 V, and scan speed 0.5−1.5 Hz. The topographic images were recorded with a resolution of 512 × 512 pixels, at which the morphology brightness increased with height. 2.3. Experimental Animals. This animal study was performed with the approval of the Animal Care and Use Committee by the Sichuan University, and special care was also taken to reduce pain to the animals. Twenty-one New Zealand rabbits that weighed approximately 2.5 kg, which were obtained from the Center of Laboratory Animals in the Clinical Medicine School at Sichuan University, were used in this study. The animals were fed a standard diet ad libitum and were housed individually under controlled temperature (22 ± 1 °C) and lighting (12 h light and 12 h darkness) conditions. With the exception of water, all food was withheld from the animals for 24 h before surgery. All of the experimental surgical procedures were performed in a sterile environment, which is in compliance with the Animal Welfare Act and the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals (NIH publication 85-23, rev. 1985). An intravenous (IV) injection of sodium pentobarbital, at a dose of 35 mg/kg, was administered as a general anesthetic. Electric clippers were used to remove the fur surrounding the intended surgical area. The exposed surgical area was wiped with povidone iodine, followed by 75% alcohol, and then surrounded with sterile-operation fabrics. All of the postoperative animals were given 0.2% ciprofloxacin at a dosage of 4 mg/kg body weight, twice a day for 3 days through intramuscular injection. The animals were

Figure 1. Schematic representation of in vivo bicortical defect in the ilium (A) and muscle implantation (B) in the paravertebral muscle of a six-month-old New Zealand rabbit.

Based on the definition for the critical size of a bony defect,26 round-shaped ilium defects were produced as follows: two holes (4 mm in diameter and 1 cm apart from each other) were drilled into the left and right ilium using a low-speed cordless drill (9.6 V, 350 rpm) equipped with a 4 mm operation aiguille. The holes were drilled through the outside plates of the ilium to a depth of approximately 0.3 cm. The holes were irrigated using a 0.9% NaCl solution at 4 °C to minimize thermal damage during drilling, and the bone debris was removed at the same time. For each rabbit, there were four bone defect holes available, two of which were randomly assigned to the RADA16-I group, one was assigned to the bone wax group, and the other to the saline group. The bone defects were lightly pressed with sterilized tampons for 30 s, and then the hemostatic materials, RADA16-I, bone wax, or the control (saline), were instantly implanted by press fitting into the bone defect holes. Some preweighed, sterilized dry-cotton tampons were used to absorb blood from the treated bone defects to prevent blood loss during the first 5 min, then they were removed, and new tampons were applied for the second 5 min. Meanwhile, the bleeding intensity was also recorded using the graded standard where 0 = none, 1 = minimal, 2 = mild, 3 = moderate, and 4 = marked.27 At the end of the experiment, absorbable 4-0 sutures were used to reapproximate the muscle and subcutaneous tissue layer, and nylon 3-0 sutures were used to close the skin. All of the postoperative animals were given 0.2% ciprofloxacin at a dose of 4 mg/kg of body weight, twice a day for 3 days. Six New Zealand rabbits were euthanized using an overdose of sodium pentobarbital at 4, 8, and 12 weeks after operation. The ilium samples were harvested for plain X-radiographs (48 kV, 100 mA, 45 or 50 ms) and histological examination. The samples were fixed in 4% polyformaldehyde, decalcified in ethylene diamine tetraacetic acid (EDTA) for 2 months, dehydrated through graded alcohol series, cleared in xylene, cut longitudinally into two halves and embedded in paraffin wax. B

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Biomacromolecules Serial sections, which had a thickness of 5 μm, were made and stained with hematoxylin−eosin (H&E) and Masson’s trichrome. 2.5. In Vivo Tissue Compatibility Test. Three 6 month old New Zealand rabbits were used to examine the compatibility of RADA16-I during the tissue compatibility test. The surgical procedure was performed according to a recently presented method:28 a 10 cm longitudinal incision was made in the middle of the dorsal fur; the subcutaneous tissue was then detached to expose the paravertebral muscle. Four muscle pockets, approximately 0.7 cm in diameter and 1.0 cm deep with 2.5 cm spacing between two bordering surgical sites, were made in the midline incisions of each paravertebral muscle using a hemostat as RADA16-I or bone wax. RADA16-I and bone wax were implanted into the four muscle pockets of the right and left paravertebral muscles, respectively. Four 100 μL aliquots of the RADA16-I stock solutions (1% w/v) were individually injected into each of the four muscle pockets. Four balls of bone wax, with a diameter of 0.3 cm, were also inserted into four other muscle pockets. All of the muscle pockets were stapled using silk sutures, which were also used to mark the implantation sites. Nylon 0-0 sutures were used to close the subcutaneous tissue and skin (Figure 1B). The animals were euthanized using an overdose of sodium pentobarbital. The paravertebral muscles were dissected, fixed in 4% polyformaldehyde, dehydrated in a graded series of ethanol, and embedded in paraffin wax. Serial sections, with a thickness of 5 μm, were cut and then stained with hematoxylin−eosin (H&E) for evaluation using light microscopy. 2.6. Statistical Analysis. All quantitative data were shown at the confidence interval x ± s (mean ± standard deviation × σ/√n), except for the local labeling. The confidence level is 95% (σ = 0.05). The quantity of sample is represented by n. The mean was analyzed for significance using the two-tailed t test.

Figure 2. Typical AFM morphological images and hydrogel formation of self-assembling peptide RADA16-I, two large field images (A, C) and higher magnification of the image inside the black frame (B). Peptide RADA16-I (100 μL, 0.17 mg/mL) self-assembles into nanofibers. Peptide RADA16-I stock solution (1% w/v) was induced to form blood−hydrogel (D) by the blood from rabbit middle auricular artery.

3. RESULTS 3.1. Self-Assembling Nanofibers and Hydrogel Formation of Peptide RADA16-I. Figure 2 shows the AFM images of the nanostructures that were formed from the RADA16-I and the formed hydrogel that was induced by the blood from the rabbit middle auricular artery. These AFM images demonstrated that peptide RADA16-I pocesses the ability of self-assemble to nanofibers (Figure 2A,C). The peptide RADA16-I exhibits an immediate morphological change from a running liquid into a red jelly-like half-solid state, which is inititated by blood through a metal ion (such as Na+ or K+) salt bridge. (Figure 2D). This formed threedimensional (3D) hydrogel, usually called the “blood−hydrogel” (Figure 2D), is constructed by interwoven nanofibers with approximately 100 nm mesh pores (Figure 2B). The RADA16-I stock solution (1% w/v) was induced to form the hydrogel from the blood of the rabbit’s middle auricular artery. A transverse cut was made in the vessel of the middle auricular artery using a scalpel blade to produce bleeding. After 10 s, 50 μL of the RADA16-I stock solution (1% w/v) was injected into the wound. As previously reported, the peptide solution formed the blood−hydrogel in approximately 10 s (Figure 2D).22,29,30 3.2. Hemostasis in an Ilium Bone Defect. Figure 3 shows the typical hemostasis in a cancellous ilium bone defect of a New Zealand rabbit that was treated with different

Figure 3. Hemostasis in an ilium bone defect of New Zealand Rabbit. The typical 4 mm diameter round-shaped ilium defects (marked with dot circles) were treated with (A) bone wax (left) and peptide RADA16-I (right) or (B) peptide RADA16-I (left) and the untreated blank (right).

hemostatic agents. A 100 μL aliquot of the RADA16-I stock solution (1% w/v) was injected into a 4 mm diameter roundshaped ilium defect. The blood−hydrogel was quickly formed, and hemostasis was achieved in approximately 10 s (Figure 3A (right),B (left)). The blood loss of “total 10 min” in the RADA16-I treatment group was approximately 0.33 g, and the bleeding intensity was “1 = minimal”, both of which were five times lower than that of the blank group (Figure 4). During the 10 min hemostatic treatment, the blood loss during the “first 5 min” and the “last 5 min” in the RADA16-I treatment group decreased to 0.16 and 0.17 g, respectively, while those in the untreated group were 0.72 and 0.92 g. There was a small amount of errhysis during the “first 5 min” and the “last 5 min” in the bone wax group, which was unable to be weighed. These results indicate that the RADA16-I treatment can achieve hemostasis and it is close to the conventional bone wax treatment with little bleeding. 3.3. Radiographic Analysis of Bone Regeneration. Figure 5 shows the bone regeneration of the ilium bone defect C

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Figure 6. Histological examination with hematoxylin and eosin (HE) staining of the ilium bone defects (vertical section) of New Zealand rabbits at different times (4, 8, and 12 weeks), when treated with 1% self-assembling peptide RADA16-I (A, B, and C), and conventional bone wax (D, E and F). The dotted lines highlight the interface between osteoid matrix and bone tissue, and the inset is the local magnification of its corresponding small black frame. The different magnifications of slides are marked at their right lower corners.

Figure 4. Blood loss of the peptide treated group and the untreated group at different times, p < 0.0001 (***).

Figure 7. Histological examination with Masson staining of the ilium bone defects (vertical section) of New Zealand rabbits at different times (4, 8, and 12 weeks), when treated with 1% self-assembling peptide RADA16-I (A, B, and C), and conventional bone wax (D, E, and F). Peptide was slightly stained (A) versus white bone wax because of its common ingredients with collagen. Similar markers, such dotted lines and arrows in Figure 6, can be used as reference here, because the same slides were used.

Figure 5. Representative radiographs of the ilium bone defects of New Zealand rabbits at different times: (A) 4 weeks, (B) 8 weeks, and (C) 12 weeks after treated with some hemostatic agents: peptide RADA16I (dotted circle), bone wax (rectangle), and the untreated blank (ellipse).

in New Zealand rabbits with different treatments. When treated with RADA16-I, the ring edges of the ilium bone defects became increasingly dim and could not be easily distinguished from their backgrounds (the dotted circles in Figure 5) at 4, 8, and 12 weeks after surgery, which suggested that they were evenly filled with newly formed bone. In contrast, the ring edge was still obvious in the bone wax treatment group (the rectangles in the lower panels of Figure 5), which reflected persistent defects at the implantation sites at 8 weeks after surgery, and at 12 weeks postoperation. Little bone healing was observed in the untreated sites (the ellipses in the upper panels of Figure 5). 3.4. Histological Examination for Bone Regeneration. Histological examination (Figures 6, 7, and 8) showed that the peptide nanofiber hydrogel did not impair osteogenesis, supported osteoblast growth, and furthermore accelerated bone regeneration. At week 4 postsurgery, the osteoblasts around the bone tissue surface (Figure 6A, black dotted line, and Figure 8A, red arrows) were flourishing and moving toward the peptide-

Figure 8. Histological examination with hematoxylin and eosin (H&E, left) and Masson (right) staining of the ilium bone defects (vertical section) of New Zealand rabbits at 4 weeks, when treated with 1% selfassembling peptide RADA16-I (A, B), and the blank (C, D), original magnification, × 100. D

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that RADA16-I can form a stable β-pleated sheet structure in water in the presence of monovalent cations such as Na+ or K+ or under physiological conditions.31,32 It can spontaneously self-assemble into a nanofiber scaffold, which consists of interwoven nanofibers with approximately 100 nm mesh pores.29,33−36 When injected into the injured site, the RADA16-I solution rapidly changed from a running liquid into a red jelly like half-solid state (called the blood−hydrogel) and covered the bleeding site immediately. The blood− hydrogel is composed of interwoven nanofibers (∼10 nm in fiber diameter) with ∼100 nm mesh pores and over 99% water. So the blood−hydrogel could arrest platelets, resulting in increased hemostasis (Figure 2D). It is believed that the inorganic salts in blood, such as Na+ and K+ ions, promote the formation of a cross-linked gel-network through the salt-bridge bond.37 The Na+ and K+ ions also play similar roles for bone tissue defects, especially for cancellous ilium bone with abundant blood sinusoids in its marrow. Meanwhile, the RADA16-I solution has an interwoven nanofiber scaffold, which is similar to naturally occurring extracellular matrix (ECM). When implanted into the ilium bone defects, RADA16-I easily filled the interconnecting labyrinthine spaces of the network of bony trabeculae, which facilitated hemostasis and supported bone cell regeneration and migration to the bone defect, resulting in accelerated osteosis. On the other hand, the selfassembling peptide hydrogels are biocompatible materials, and autodegrade in vivo under condition with high concentration, resulting in inflammation-free response. However, because of weakness of mechanical strength of the peptide hydrogel, it was unable to fully withstand the pressure of bleeding, and there was still a little blood leakage during the 10 min hemostatic treatment. A problem associated with using bone wax as a hemostatic agent is that bone wax is too tough, which prevents the penetration of osteoblasts into this material, and it may form a physical barrier that prevents the reunion of bony surfaces (Figure 6F and 7F). In contrast, the RADA16-I hydrogel contains 99% water and thus can be delivered as a liquid material. This porous material supplies a true 3-D environment for cell growth, differentiation, and migration.38,39 For example, RADA16-I can promote the differentiation of adult progenitor cells into functional hepatocyte-like spheroid clusters21 and facilitate bone regeneration for supporting the penetration and engrafting of cells in calvaria bone defects in mice.24,40 Moreover, the soft peptide can enable enzymatic degradation by matrix metalloproteinases and aggrecanases that are naturally synthesized by cells,16 and subsequently results in the appearance of bony bridges. The effects of the RADA16-I hydrogel and bone wax on the regeneration of bone tissue were comparatively examined in an ilium bone defect model in New Zealand rabbits. On the one hand, there was an obvious migration of osteoblasts into the defect area, followed by the formation of mature bone tissues. On the other hand, no obvious bone regeneration was observed in the bone defect area that was treated with bone wax. Meanwhile, we also noticed that not only bone wax but also other commercially available hemostatic agents were widely used for controlling bleeding from bony structures. A similar examination will perform in our next study. In addition to the effectiveness and resorbability, the RADA16-I hydrogel also has good tissue compatibility, which is very important as a hemostatic agent. In the RADA16-I hydrogel treatment group, few foreign body giant cells and

treated defect sites without any barrier, leading to obvious reduction of the defect area compared with that in the bone wax treatment group (Figures 6D and 7D, the white area). There was no evident interface between the peptide hydrogel and the osteoid matrix (Figure 6A, the right upper part of the inset, Figure 8A,B, red arrows), whereas a clear interface and inflammatory cells can be observed in the bone wax treatment group (Figure 6D, the right side of the inset) and in the blank group (Figure 8C,D, black arrows), suggesting that the peptide nanofiber scaffolds can support osteoblast transfer and growth. At week 8 postsurgery, the peptide-treated defects were fully filled with osteoblasts, osteoid, and newly formed bone matrix (Figures 6B and 7B); furthermore, bone trabecula and bone reconstruction occurred in these defects (Figure 6B, arrow). Meanwhile, the bone wax-treated defects were still filled with abundant bone wax (Figures 6E and 7E). At week 12 postsurgery, newly formed bone was observed in the center of the bone defects that were treated with RADA16-I hydrogel, and the defects possessed medullary cavities inside of the ilium bone (Figure 6C, black dotted line and arrow, Figure 7C), thus indicating regeneration of the mature bone tissue. On the other hand, no obvious bone regeneration occurred in the bone defects that were treated with bone wax (Figures 6F and 7F). 3.5. In Vivo Tissue Compatibility Examination. H&E staining indicated that the RADA16-I hydrogel was almost free of inflammatory reactions; only a few foreign body giant cells were observed surrounding the implanted site (Figure 9A, red

Figure 9. Histological examination with hematoxylin and eosin (H&E) staining of the paravertebral muscle of a six-month-old New Zealand rabbit at different times (2, 4, and 8 weeks), when implanted with 1% self-assembling peptide RADA16-I (A, B, and C), and conventional bone wax (D, E, and F). The dotted line (F) highlights the interface between new muscle tissue and fibrous encaspsulation, and the inset is the local magnification of its corresponding small black frame, original magnification, ×100.

star) at 2 weeks after surgery. At 4 weeks, new muscle tissue (Figure 9B) and fibroblasts (Figure 9B, the inset, thin arrows) occupied the injured sites, and at 8 weeks new muscle tissue was formed (Figure 9C). In contrast, after 8 weeks, a moderate inflammatory response and vigorous foreign body giant cells were found at the interface of the bone wax (Figure 9D−F, thick arrows) and the surrounding tissues (Figures 9D−F, black stars and dotted line). These data indicate the good biological tissue compatibility of RADA16-I.

4. DISCUSSION In the present work, we focused on the application of RADA16I hydrogel for hemostasis induction. It has been well established E

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(11) Zhang, S.; Zhao, X. J. Mater. Chem. 2004, 14 (14), 2082−2086. (12) Hosseinkhani, H.; Hong, P.-D.; Yu, D.-S. Chem. Rev. 2013, 113, 4837−4861. (13) Cheng, T.-Y.; Wu, H.-C.; Huang, M.-Y.; Chang, W.-H.; Lee, C.H.; Wang, T.-W. Nanoscale 2013, 5 (7), 2734−2744. (14) Soler-Botija, C.; Bago, J. R.; Llucia-Valldeperas, A.; Valles-Lluch, A.; Castells-Sala, C.; Martinez-Ramos, C.; Fernandez-Muinos, T.; Chachques, J. C.; Pradas, M. M.; Semino, C. E.; Bayes-Genis, A. Am. J. Transl. Res. 2014, 6 (3), 291−301. (15) Ellis-Behnke, R. G.; Liang, Y. X.; You, S. W.; Tay, D. K.; Zhang, S.; So, K. F.; Schneider, G. E. Proc. Natl. Acad. Sci. U. S. A. 2006, 103 (13), 5054−9. (16) Holmes, T. C.; de Lacalle, S.; Su, X.; Liu, G.; Rich, A.; Zhang, S. Proc. Natl. Acad. Sci. U. S. A. 2000, 97 (12), 6728−33. (17) Mi, K.; Wang, G.; Liu, Z.; Feng, Z.; Huang, B.; Zhao, X. Macromol. Biosci. 2009, 9 (5), 437−43. (18) Bokhari, M. A.; Akay, G.; Zhang, S.; Birch, M. A. Biomaterials 2005, 26 (25), 5198−208. (19) Semino, C. E.; Kasahara, J.; Hayashi, Y.; Zhang, S. Tissue Eng. 2004, 10 (3−4), 643−55. (20) Nowakowski, G. S.; Dooner, M. S.; Valinski, H. M.; Mihaliak, A. M.; Quesenberry, P. J.; Becker, P. S. Stem Cells 2004, 22 (6), 1030−8. (21) Semino, C. E.; Merok, J. R.; Crane, G. G.; Panagiotakos, G.; Zhang, S. Differentiation 2003, 71 (4−5), 262−70. (22) Ellis-Behnke, R. G.; Liang, Y. X.; Tay, D. K.; Kau, P. W.; Schneider, G. E.; Zhang, S.; Wu, W.; So, K. F. Nanomedicine 2006, 2 (4), 207−15. (23) Misawa, H.; Kobayashi, N.; Soto-Gutierrez, A.; Chen, Y.; Yoshida, A.; Rivas-Carrillo, J. D.; Navarro-Alvarez, N.; Tanaka, K.; Miki, A.; Takei, J.; Ueda, T.; Tanaka, M.; Endo, H.; Tanaka, N.; Ozaki, T. Cell Transplant 2006, 15 (10), 903−10. (24) Liu, J.; Zhao, X. Nanomedicine (London, U. K.) 2011, 6 (9), 1621−43. (25) Gogolewski, S.; Gorna, K.; Turner, A. S. J. Biomed. Mater. Res., Part A 2006, 77 (4), 802−10. (26) Schmitz, J. P.; Hollinger, J. O. Clin. Orthop. Relat. Res. 1986, No. 205, 299−308. (27) Bertone, A.; Lipson, D.; Kamei, J.; Litsky, A.; Weisbrode, S. Clin. Orthop. Relat. Res. 2006, 446, 278−85. (28) Wellisz, T.; Armstrong, J. K.; Cambridge, J.; Fisher, T. C. J. Craniofac. Surg. 2006, 17 (3), 420−5. (29) Ye, Z.; Zhang, H.; Luo, H.; Wang, S.; Zhou, Q.; Du, X.; Tang, C.; Chen, L.; Liu, J.; Shi, Y. K.; Zhang, E. Y.; Ellis-Behnke, R.; Zhao, X. J. Pept. Sci. 2008, 14 (2), 152−62. (30) Kopecek, J.; Yang, J. Acta Biomater. 2009, 5 (3), 805−16. (31) Cormier, A. R.; Pang, X.; Zimmerman, M. I.; Zhou, H. X.; Paravastu, A. K. ACS Nano 2013, 7 (9), 7562−72. (32) Ravichandran, R.; Griffith, M.; Phopase, J. J. Mater. Chem. B 2014, 2, 8466−78. (33) Zhang, S.; Holmes, T.; Lockshin, C.; Rich, A. Proc. Natl. Acad. Sci. U. S. A. 1993, 90 (8), 3334−8. (34) Zhang, S.; Lockshin, C.; Cook, R.; Rich, A. Biopolymers 1994, 34 (5), 663−72. (35) Zhang, S.; Rich, A. Proc. Natl. Acad. Sci. U. S. A. 1997, 94 (1), 23−8. (36) Altman, M.; Lee, P.; Rich, A.; Zhang, S. Protein Sci. 2000, 9 (6), 1095−105. (37) Fichman, G.; Gazit, E. Acta Biomater. 2014, 10, 1671. (38) Gelain, F.; Bottai, D.; Vescovi, A.; Zhang, S. PLoS One 2006, 1 (1), e119. (39) Wu, M.; Ye, Z. Y.; Liu, Y. F.; Liu, B.; Zhao, X. J. J. Nanomater. 2010, 2010, 437219. (40) Firth, A.; Aggeli, A.; Burke, J. L.; Yang, X.; Kirkham, J. Nanomedicine (London, U. K.) 2006, 1 (2), 189−99. (41) Liedmann, A.; Rolf, A.; Frech, M. J. J. Visualized Exp. 2012, 59, e3830. (42) Akiyama, N.; Yamamoto-Fukuda, T.; Takahashi, H.; Koji, T. Int. J. Nanomed. 2013, 8, 2629−40.

inflammatory reactions were observed after surgery. Cytotoxicity associated with the low pH of RADA16-I has been reported in recent years.41−47 To avoid cell necrosis and receive the full benefit of the self-assembling peptide technology, a preneutralization procedure was often employed,48,49 but no particular preprocess was performed in this study, so that the low viscosity RADA16-I can fully immerse into the injured bony tissue, and the remnant saline was employed for neutralization and gelatinization of RADA16-I peptide. Therefore, compared with bone wax, the designed peptide to tamponade a hemorrhage can distinctly decrease the risk from toxicity and complications.49

5. CONCLUSIONS RADA16-I can self-assemble into a nanofiber hydrogel and thus can be used as an effective hemostatic agent. Its physical properties and usability were better than conventional bone wax. A hemostasis experiment in an ilium bone defect indicated that the RADA16-I hydrogel on bone tissue induced hemostasis similarly to the effects observed using bone wax. In all of the present indication examinations, it was observed that the RADA16-I hydrogel has significant advantages over bone wax. For example, the RADA16-I hydrogel permits the penetration of osteoblasts, allowing them to settle deeply into the defect area and facilitate bone regeneration. In addition, high concentration RADA16-I is fairly free from the risk of toxicity and complications. Finally, because of the good hydrophilic nature, the RADA16-I hydrogel could serve an additional purpose by acting as a carrier for the prolonged delivery of growth factors, antibiotics, or drugs to the damaged bone.

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AUTHOR INFORMATION

Corresponding Authors

*Fax: +86-28-8516-4073. Tel: +86-28-8516-4069. E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank all members of their laboratory. This work was partly supported by the Chinese National 985 Project of Education Ministry to Sichuan University and by the National Natural Science Foundation of China (Grant No. 81371536, 81501462).

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DOI: 10.1021/acs.biomac.5b00493 Biomacromolecules XXXX, XXX, XXX−XXX