b-poly(ethylene glycol)-b-poly(ε-caprolactone) Porous Microspheres

Feb 6, 2018 - Then, a critical granulation tissue consisted of inflammatory cells, fibroblast cells, and invasion capillaries. Figure 4. CLSM images o...
0 downloads 0 Views 2MB Size
Download PDF
Article Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Injectable Hybrid Poly(ε-caprolactone)‑b‑poly(ethylene glycol)‑b‑poly(ε-caprolactone) Porous Microspheres/Alginate Hydrogel Cross-linked by Calcium Gluconate Crystals Deposited in the Pores of Microspheres Improved Skin Wound Healing JinFeng Liao,†,‡,# YanPeng Jia,†,# BeiYu Wang,† Kun Shi,† and ZhiYong Qian*,† †

State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu 610041, People’s Republic of China ‡ State Key Laboratory of Oral Diseases, West China School of Stomatology, Sichuan University, Chengdu 610041, People’s Republic of China ABSTRACT: In our study, a hybrid alginate hydrogel crosslinked by calcium gluconate crystals deposited in poly(εcaprolactone)-b-poly(ethylene glycol)-b-poly(ε-caprolactone) (PCL-PEG-PCL, abbreviated as PCEC) porous microspheres was developed for skin engineering. The diameter of microspheres was ∼212 μm, and the pore size was ∼8 μm. The PCEC porous microspheres supplied different functions in the hydrogel: (1) Calcium gluconate crystals were loaded in the inner pores of the microspheres, which can induce alginate hydrogel to cross-link in a few minutes once they were mixed. (2) The porous structure of the microspheres provided more anchor points for fibroblast attachment and growth, resulting in the enhancement of cell growth in the hybrid hydrogel. The PCEC microspheres/Alg hydrogel (MPs/Alg hydrogel) possessed excellent compatibility, because cell viability remained around 100% even at a concentration of 500 μg/mL. Meanwhile, the morphology of 3T3 and L929 cells attached on both PCEC porous microspheres and MPs/Alg hydrogel were confirmed by confocal laser spectrometry (CLSM). What’s more, MPs/Alg hydrogel promoted wound regeneration in a full-thickness skin defect model of rats. The mild inflammation reaction existed at the early stage of wound repair and gradually disappeared. These findings suggested that MPs/ Alg hydrogel may possess great potential in the application of skin tissue engineering. KEYWORDS: porous PCEC microspheres, calcium gluconate cross-linker, injectable, alginate hydrogel, skin dressing

■

foams and hydrogels, etc.20−25 Among these wound healing materials, hydrogels can meet most of the requirements as skin substitutes and be commonly used as wound dressings due to their good viscoelasticity and water content. To overcome the wound dehydration, it is important to maintain the wound bed in a moist condition for effective skin regeneration. Because of the hydrophilic ability and porous structure, hydrogels can keep wounds in a wet status, absorb the redundant tissue excretions, and permit the transportation of oxygen or other nutrient substances.26 These advantages make hydrogel attractive among the various wound dressings. In particular, the injectable hydrogel has been a more desirable kind of wound dressing, as it is able to mold into the wound defect for matching the shape well.27,28 The injectable hydrogel was in a fluid state before use, and upon injection it converted into a hydrogel state. Thus, it filled wound sites, especially irregular regions. Although the injectable hydrogel had three-dimensional structure with numerous pores, it is difficult for cells to anchor

INTRODUCTION Trauma, burns or diabetic foot ulcers can lead to cutaneous wounds, which may cause major medical burdens.1−3 The gold standard of cutaneous wound treatment in the clinic is autologous skin grafts, which are seriously limited by the lack of a donor skin supplement and the undesirable injury of the donor region.4−6 The re-establishment of structural continuity in wound skin involved in not only the migration of keratinocytes and fibroblast toward the wound region but also the deposition of new extracellular matrix (ECM) components, which may rely on the scaffold to a large extent.7−9 Skin healing enhancement is critical to the patients’ suffering from cutaneous wounds. Therefore, suitable wound dressings play a pivotal role as a skin shield in the period of wound healing.10−14 To satisfy the requirements, an ideal skin dressing needs to possess appropriate pore size (or porosity), good biocompatibility, and good matching with the defect region to accelerate wound healing.15,16 Numerous efforts were made to design and fabricate skin scaffolds for the healing of wounds.17−19 Up to now, many kinds of skin dressings have been developed, including membranes, electrospun nanofibers, colloidal nanoparticles, © XXXX American Chemical Society

Received: November 9, 2017 Accepted: February 6, 2018 Published: February 6, 2018 A

DOI: 10.1021/acsbiomaterials.7b00860 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Article

ACS Biomaterials Science & Engineering

Scheme 1. Process of Mice with Skin Defect Treated with Injectable MPs/Alg Hydrogel and Improved Skin Regeneration

of dry copolymer was studied with a differential scanning calorimeter (DSC, NETSCZ, Germany). Porous PCEC microspheres were prepared by the method of waterin-oil-in-water (w/o/w) double emulsion with a bit change. A total of 3.75 mL of 5% (w/v) NH4HCO3 in deionized water was added to 12 mL of 6.25% (w/v) PCEC in methylene chloride. The w/o primary emulsion was performed by using a homogenizer (Powergen 700) with a speed of 5000 rpm for 3 min. Subsequently, the formed w/o emulsion was poured into 450 mL of 0.5% (w/v) PVA solution in a beaker. The w/o/w double emulsion was then prepared by mixing via an overhead propeller (LR-400A, Fisher Scientific Co., USA). After stirring at 1500 rpm for 8 h, the organic solvent (methylene chloride) was completely evaporated. The porous microspheres were obtained after being etched with NaOH. Finally, they were separated by centrifugation and washed with distilled water three times. The porous PCEC microspheres were soaked in 3% calcium gluconate solution for 3 h. Then, all the calcium gluconate solution was decanted, and EtOH was added to deposit calcium gluconate crystals in the inner pores of PCEC microspheres. The above process was repeated three times. The samples of calcium gluconate crystals loaded in PCEC microspheres were freeze-dried before use. The morphologies of the microspheres were observed with a digital image analysis system (Nikon E 600 Microscope with a Nikon Digital Camera DXM 1200, Nikon Corporation, Japan) and scanning electron microscope (SEM, JSM-5900LV, JEOL, Japan). Preparation and Characterizations of PCEC Microspheres/ Alginate Hydrogel. Stock solutions of 1.50 g of alginate in 100 mL of deionized water were prepared. Subsequently, 50 mg of PCEC microspheres loaded with calcium crystals were mixed with 0.75 mL of alginate stock solution. The mixture was homogenized to obtain a homogeneous distribution of microspheres and induce calcium ion cross-linking alginate to form microspheres/alginate hydrogel (MPs/ Alg hydrogel). The MPs/Alg hydrogel was lyophilized and cut to observe the cross-section morphology by SEM. Cytotoxicity Analysis Test. The cytotoxicity of hybrid MPs/Alg hydrogel was measured using 3T3 and L929 cells. The cells were grown in 96-well plates at a density of 5.0 × 103 in 100 μL of DMEM medium and incubated at 37 °C overnight for cell attachment. Then, various concentrations of MPs/Alg hydrogel were incubated with cells for 24 h. The cell viability was tested using the MTT method. Fibroblasts Growth and Morphology Studies on Both PCEC Porous Microspheres and Hybrid Microspheres/Alg Hydrogel. 3T3 and L929 cells were seeded in the porous microspheres and hybrid MPs/Alg hydrogel at a density of 1.0 × 105 and subsequently grown at 37 °C. After proliferation for 3 days, the cellular constructs were rinsed with PBS three times and fixed with 3.7% formaldehyde for 15 min. Then, 0.2% Triton-100 was used to permeabilize the cells. And the cells were stained with alexa-fluor 488 phalloidin for the actin cytoskeleton and DAPI for the nucleus.30 The morphologies of 3T3 and L929 fibroblasts on PCEC microsphere/alginate hydrogel were observed by a Leica TCS SP2 laser scanning confocal microscopy

themselves on the smooth surface of these pores. Thus, we introduced a kind of porous microsphere made of poly(εcaprolactone)-b-poly(ethylene glycol)-b-poly(ε-caprolactone) (PCL-PEG-PCL, abbreviated as PCEC) copolymer with a rough surface to favor the cell attachment and growth. εCaprolactone was used to adjust the hydrophilic ability of PEG. In this way, the microspheres made by PCEC possess the hydrophobic property for better cell attachment. The PCEC porous microspheres first deposited calcium gluconate crystals in their inner pores, which played the role of forming injectable cross-linked alginate hydrogel. As shown in Scheme 1, the process of treating mice with skin defects with injectable PCEC microspheres/alginate (MPs/Alg) hydrogel had significantly improved skin regeneration with a recovery skin structure. With more useful anchor sites supplied by porous microspheres, the hybrid hydrogel helped improve the cells’ adhesion and proliferation, leading to a better skin regeneration in the wound defect.

■

MATERIALS AND METHODS

Materials. Poly(ethylene glycol) (PEG, Mn = 4000), εcaprolactone (ε-CL), stannous octoate (Sn(Oct)2), alexa-fluor 488 phalloidin, and poly(vinyl alcohol) (PVA, Average Mn = 30 000− 70 000) were obtained from Sigma-Aldrich Company, USA. Calcium D-gluconate monohydrate was purchased from Aladdin Industrial Corp., Shanghai, China. Alginate, ammonium bicarbonate (NH4HCO3), sodium hydroxide, dichloromethane (DCM), ethanol (EtOH), and phosphate buffer saline (PBS) were acquired from Kelong Chemicals, Chengdu, China. Monohydrate 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) was purchased from Roche, USA. For the in vivo skin defect study, 12 healthy Sprague−Dawley (SD) rats (male, 200−220 g) were obtained from Beijing HFK Bioscience Co., Ltd. They were kept for an adaptive period of 1 week before surgery. All the marine operation procedures were approved by the animal care and use committee of State Key Laboratory of Biotherapy, Sichuan University. Throughout the experimental period, the animals were treated humanely. Preparation and Characterizations of Porous PCEC Microspheres and Calcium Gluconate Deposited in PCEC Microspheres. According to the previous study,29 the PCEC (Mn = 50 kDa) three block copolymer was synthesized by ring-opening polymerization of poly(ethylene glycol) (PEG, Mn = 4000) and εcaprolactone (ε-CL) catalyzed by Sn(Oct)2 at 130 °C. After purification, the freeze-dried PCEC product was characterized by 1 H-nuclear magnetic resonance spectroscopy (1H NMR, Varian 400 spectrometer, Varian, USA) and Fourier transform infrared (FT-IR, Nicolet FTIR spectrometer, Thermo Scientific). The thermal behavior B

DOI: 10.1021/acsbiomaterials.7b00860 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Article

ACS Biomaterials Science & Engineering

Figure 1. (A) 1H NMR and (B) FT-IR spectra of PCEC polymer. (C) The DSC analysis curves of PCEC polymer.

melting property of ε-CL.33 The peak of PEG was covered by the PCL, because the majority proportion of polymer was εCL.29 Subsequently, the porous PCEC microspheres were prepared via the method of w/o/w double emulsion-solvent evaporation. And the morphologies of microspheres and their porous structure can be seen from the microscope image, as shown in Figure 2A. The diameter of microspheres was ∼212 μm and pore size was ∼8 μm. Because of the porous structure, the light can penetrate through the microspheres. When porous microspheres soaked in ethanol first, the calcium gluconate crystals were loaded over the shell of microspheres (Figure 2B). On the contrary, the porous microspheres soaked in calcium gluconate solution first; calcium crystals were deposited in the inner pores of PCEC microspheres (Figure 2C and D). From the microscope image of Figure 2C, some blank spots can be seen in the inner section of microspheres, resulting in weakening the light penetration. The process and quantification analysis of loaded calcium crystals can be found in our previous research work in detail.34 The obtained calcium loaded PCEC microspheres were then mixed with alginate solution to form an injectable hydrogel, which can be easily extruded through an injection spring with an 18G gauge needle (Figure 2F). After around 3 min, the cross-linked PCEC porous microspheres/ alginate hybrid hydrogel can fix into a designed form, as shown in Figure 2G. The freeze-dried sample was used to observe the cross-section morphology features via SEM. From Figure 2E, the hydrogel showed a porous structure, and the PCEC microspheres inlaid in the pores of hydrogel. Cytotoxicity Analysis Test of Microspheres/Alg Hydrogel and Cell Attachment/Proliferation Analysis on PCEC Microspheres. Good cell compatibility is the vital property of biomaterials for further study. Thus, the

(Leica Microsystem Inc., Germany). In addition, the morphologies of fibroblasts on PCEC microspheres and PCEC microspheres/alginate hydrogel were also visualized by SEM. CLSM images of PCEC porous microspheres after L929 fibroblast cultivation for 1 day, 3 days, and 7 days were studied. Skin Regeneration Experiment in SD Rats. A total of 12 healthy eight-week-old male SD rats (200−220 g) were anaesthetized by 10% chloral hydrate. The dorsal hair was removed by using a shaving machine. Then, the surgical regions were disinfected with iodine and 75% ethanol. A full-thickness skin defect with a length and width of 1.0 cm was created by using iris scissors on the dorsal area of each rat. The PCEC microsphere/Alg hydrogel dressing was injected to cover the defect section. The control group was not treated with any materials. Finally, the surgical areas in both groups were covered with the application. The rats after surgery were raised in individual cages to prevent attack or fighting on their wound sites. At 7 and 14 days after surgery, the morphology of the wound was examined and photographs were taken. Then, the rats were sacrificed to remove the wound area together with the surrounding tissues for further study. Histological Study. The harvested skin samples were fixed in 4% paraformaldehyde (in 0.1 M PBS, pH = 7.4) for 2 days. Subsequently, they were dehydrated with a series of graded ethanol and mounted into paraffin. After dewaxing, tissue sections were cut to 5 μm thickness for H&E and Masson’s trichrome staining.

■

RESULTS AND DISCUSSION The Preparation and Characterizations of PCEC Microspheres/Alginate Hydrogel. First, we synthesized PCEC (Mn = 50 kDa) by ring-opening polymerization of εCL and PEG4000. The 1H NMR (Figure 1A) and FT-IR spectra (Figure 1B) show that the PCEC copolymer was successfully synthesized, according to our groups’ previous reports.31,32 Meanwhile, the DSC of PCEC microspheres was also characterized, as shown in Figure 1C. The DSC curve exhibited a melting transition peak at 56 °C under a heating process and 34 °C under a cooling process, contributing to the C

DOI: 10.1021/acsbiomaterials.7b00860 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Article

ACS Biomaterials Science & Engineering

around 100% even at a material concentration of 500 μg/mL. And the cell viability had no significant difference when the concentration of PCEC microspheres/Alg hydrogel ranged from 0 to 500 μg/mL. The results suggest that the PCEC microspheres/Alg hydrogel was compatible with cells. To study the ability of the microsphere and hybrid hydrogel as cell carriers, the research on cell attachment and proliferation was investigated on PCEC microspheres first. The 3T3 and L929 cells were grown onto prewetted porous microspheres in designed time. After incubation for 3 days, the cell attachments were assessed by SEM and confocal laser scanning microscopy (CLSM). Figure 3C and D showed that the 3T3 and L929 cells were attached on the surface of microspheres, and some cells have crossed the pores of the microsphere to form as bridges. Meanwhile, Figure 4A, B, and C displayed confocal images of L929 fibroblasts with their nucleus stained by DAPI after being seeded onto microspheres. The number of L929 cells increased within the culture period. Furthermore, the infiltration of L929 cells into the porous microspheres was studied at 7 days after cultivation. As shown in Figure 4D, the fluorescence images were taken at the same x and y axis, but different z positions across the thickness of microspheres. The L929 cells not only adhered to the outer surface region of microspheres but also appeared to have infiltrated the inner pores of the microspheres at different depths below the surface after 7 days’ cultivation. The results of high efficiency on the fibroblasts’ attachment and proliferation onto the PCEC microspheres may contribute to the highly porous structure of microspheres, which lead to a significantly high surface area. Meanwhile, the surface of the microspheres was hydrophobic and hydrophilic due to the three blocks copolymer. The crude surface may also absorb more cells to adhere to the microspheres. Thus, the porous

Figure 2. Microscope images of (A) PCEC porous microspheres, (B) PCEC porous microspheres loaded with calcium gluconate crystals over the shell and (C) PCEC porous microspheres deposited with calcium gluconate crystals in the inner pores. (D) SEM images of PCEC porous microspheres loaded with calcium gluconate crystals in the pores. (E) SEM image of the cross-section of PCEC porous microspheres/alginate hydrogels. (F) The injectable PCEC porous microspheres/alginate hybrid hydrogel though the syringe. (G) The gross morphology of the cross-linked microspheres/alginate hybrid hydrogel.

cytotoxicity of PCEC microspheres/Alg hydrogel was studied first. As shown in Figure 3A and B, cell viability remained

Figure 3. Cytotoxicity analysis of PCEC microspheres/Alg hydrogel on (A) 3T3 cells and (B) L929 cells. SEM photographs of (C) 3T3 cells and (D) L929 cells cultured on porous PCEC microspheres for 3 days. D

DOI: 10.1021/acsbiomaterials.7b00860 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Article

ACS Biomaterials Science & Engineering

Figure 4. CLSM images of PCEC porous microspheres after L929 fibroblasts cultivation for (A) 1 day, (B) 3 days, (C) 7 days. (D) CLSM images of L929 fibroblasts on porous PCEC microspheres for 7 days. Serial optical slices were obtained along microspheres’ vertical z-axis, and the number on the right top of each lattice denotes the coordinate of the slice on the z-axis.

microspheres with a rough surface can attract cell adhesion proteins, such as vitronectin and fibronectin at significantly higher levels than the solid microspheres with a smooth surface.35 Cell Morphology on PCEC Microspheres/Alginate Hydrogel. The vital factors of scaffold design in tissue engineering not only require use of biocompatible materials but also supply enough space for cell seeding and proliferation. The scaffold also plays an important role in the cell morphology after they attached to the scaffold. SEM images showed 3T3 cells (Figure 5A) and L929 cells (Figure 5B) coimmobilized in PCEC porous microspheres/alginate hydrogels after incubation for 3 days. The cells spread well in the scaffold. Then, fluorescence imaging via CLSM was used to further study the cell skeleton morphologies of 3T3 (Figure 5C) and L929 (Figure 5D) fibroblasts on PCEC microspheres/alginate hydrogel. FITC-conjugated phalloidin and DAPI were used to stain the cytoskeletal protein actin and cell nucleus, respectively. Under CLSM, it is observed that 3T3 and L929 fibroblasts developed an extended dendritic morphology on the MPs/Alg hydrogel. This result further revealed that successful attachment and good spread of the fibroblasts in the scaffold, which suggested potential applications of microsphere-alginate hydrogel in cell encapsulation and skin tissue regeneration. Skin Regeneration by PCEC Microspheres/Alginate Hydrogel. The in vivo skin defect regeneration research was studied by using a full-thickness skin defect model on SD rats. The length and width of the wound defect were both 1.0 cm. In one group, the skin wounds were covered with the MPs/Alg hydrogel, and the other group without treatment was set as a control group (Figure 6). The wounds in both groups were finally covered with applications. The wounds treated with MPs/Alg hydrogel exhibited dryness on the wound region. Meanwhile, no pathological fluid oozed out at any time. No

Figure 5. SEM images and confocal microscopy images of (A, C) 3T3 cells and (B, D) L929 cells coimmobilized in PCEC porous microspheres/alginate hydrogels (scale bars in (C) and (D) = 1 μm). Cells stained with 4′,6-diamidino-2-phenylindole (DAPI, nuclei) and FITC-conjugated phalloidin (f-actin).

significantly inflammatory reaction or infection condition appeared in the wounds of the hybrid hydrogel group compared with control group during the regeneration period. After 7 days’ treatment, the MPs/Alg hydrogel treated group showed better skin recovery than the control group, as shown in Figure 6. At 14 days, the wounds treated with MPs/Alg hydrogel exhibited an almost fully recovered skin, whereas those untreated showed an incomplete recovery. The process of skin repair first formed a clot, which filled out the wound area. Then, a critical granulation tissue consisted of inflammatory cells, fibroblast cells, and invasion capillaries E

DOI: 10.1021/acsbiomaterials.7b00860 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Article

ACS Biomaterials Science & Engineering

activity than the control group. On the 14th days, there was no obvious difference in the wound closure between the two groups, but their score in granulation tissue and collagen deposition showed a big difference. The granulation tissue score in the MPs/Alg hydrogel treated group was 4.17, which was higher than 3.57 for the control group. Overall, the wounds treated with MPs/Alg hydrogel not only possessed more granulation tissue, but also a higher degree of fibroblastic deposition and re-epithelialization compared with the control group. The significant difference in wound closure and epidermis thickness between the two groups can be found on the day 7, indicating a positive influence of the scaffold treatment on the defect healing in the earlier phase. The better results of granulation tissue and collagen deposition in two phases of the healing indicated that the MPs/Alg hydrogel enhanced the function recovery of skin. The histological analysis was further performed for the wound healing. The H&E staining was displayed in Figure 8A.

Figure 6. Macroscopic appearance of skin wounds in the control group and PCEC microspheres/Alg hydrogel group at 0 days, 7 days, and 14 days after surgery.

formed.36 Meanwhile, the epidermal tissue regeneration covered the denuded wound surface. Thus, it is necessary for us to evaluate quantitatively the area of wound closure, epidermis thickness, granulation tissue formation, and collagen deposition, as shown in Figure 7. At day 7, the percent of

Figure 8. (A) The H&E stained sections and (B) Masson’s trichrome staining of the granulation tissue in control and PCEC microspheres/ Alg hydrogel group at 7 days and 14 days wound healing (scale bar = 50 μm).

More smooth epidermis was formed in the wound treated with MPs/Alg hydrogel than those in the control group on postoperative day 7. The hair follicles, as the particular auxiliary organ of a completely developed skin structure, regenerated in the MPs/Alg group, which were bare in the control group at day 14. The inflammation status was also analyzed. In the superficial dermis, vertical growth of capillary vessels and infiltration of lymphocytes can be observed at day 7, which revealed the early period of skin repair in both groups. Meanwhile, fibrous hyperplasia was demonstrated by the huge increase of fibroblasts. From 7 days to 14 days, the obvious change was that inflammatory cells decreased while collagen increased. In addition, reduced lumen and scar repair also illustrated the wound healing process. Therefore, mild inflammation reaction existed at the early stage of wound healing and gradually disappeared when the wound finished healing. To investigate the collagen deposition, Masson’s trichrome staining sections were carried out at the same designed time points. As shown in Figure 8B, the collagen was stained a blue color. Collagen density increased more significantly over time in the MPs/Alg hydrogel treated group than control group. Furthermore, densely packed collagen fibers were observed in the MPs/Alg hydrogel treated wounds on day 14. And the collagen fibers with parallel arrangement showed more accumulation in the dermal regeneration region. In contrast, the control group only possessed less accumulation of collagen fibers, which appeared in a loosely packed and irregular arrangement. The results indicated a faster and more effective skin defect regeneration and function restoration in the MPs/Alg hydrogel treatment group when compared with the no treatment group.

Figure 7. (A) The wound closure, (B) thickness of epidermis, (C) granulation tissue score, and (D) collagen content of the granulation tissue in control and PCEC microspheres/Alg hydrogel group at 7 days and 14 days.

wound coverage in the MPs/Alg hydrogel treated group already reached 91%. However, the control group only exhibited 75% coverage. Meanwhile, the thickness of the epidermis in MPs/ Alg hydrogel treated group and control group were 49.7 and 23.3 μm, respectively. Granulation tissue was formed by fibroblasts, accumulated extracellular matrix, and other cells.37 The better the granulation tissue formation, the better skin defect regeneration that would appear. The granulation tissue score and collagen deposition content in the experiment group were 2.43 and 57%, which suggested a better regeneration F

DOI: 10.1021/acsbiomaterials.7b00860 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Article

ACS Biomaterials Science & Engineering

■

promotes the healing of acute skin wounds in rats. Sci. Rep. 2017, 7, 45183. (9) Zhu, F.; Wang, C.; Yang, S.; Wang, Q.; Liang, F.; Liu, C.; Qiu, D.; Qu, X.; Hu, Z.; Yang, Z. Injectable tissue adhesive composite hydrogel with fibroblasts for treating skin defects. J. Mater. Chem. B 2017, 5, 2416−2424. (10) Berthet, M.; Gauthier, Y.; Lacroix, C.; Verrier, B.; Monge, C. Nanoparticle-based dressing: the future of wound treatment. Trends Biotechnol. 2017, 35 (8), 770−784. (11) Amirian, J.; Lee, S. Y.; Lee, B. T. Designing of combined nano and microfiber network by immobilization of oxidized cellulose nanofiber on polycaprolactone fibrous scaffold. J. Biomed. Nanotechnol. 2016, 12, 1864−1875. (12) Liu, M.; Luo, G.; Wang, Y.; Xu, R.; Wang, Y.; He, W.; Tan, J.; Xing, M.; Wu, J. Nano-silver-decorated microfibrous eggshell membrane: processing cytotoxicity assessment and optimization, antibacterial activity and wound healing. Sci. Rep. 2017, 7 (1), 436. (13) Rangasamy, S.; Tak, Y. K.; Kim, S.; Song, J. M.; Paul, A. Bifunctional therapeutic high-valence silver-pyridoxine nanoparticles with proliferative and antibacterial wound-healing activities. J. Biomed. Nanotechnol. 2016, 12, 182−196. (14) Shen, Y.; Dai, L.; Li, X.; Liang, R.; Guan, G.; Zhang, Z.; Cao, W.; Liu, Z.; Mei, S.; Liang, W.; Qin, S.; Xu, J.; Chen, H. Epidermal stem cells cultured on collagen-modified chitin membrane induce in situ tissue regeneration of full-thickness skin defects in mice. PLoS One 2014, 9 (2), e87557. (15) Sun, B. K.; Siprashvili, Z.; Khavari, P. A. Advances in skin grafting and treatment of cutaneous wounds. Science 2014, 346 (6212), 941−945. (16) Vedakumari, W. S.; Prabu, P.; Sastry, T. P. Chitosan-fibrin nanocomposites as drug delivering and wound healing materials. J. Biomed. Nanotechnol. 2015, 11, 657−667. (17) Eke, G.; Mangir, N.; hasirci, N.; MacNeil, S.; Hasirci, V. Development of a UV crosslinked biodegradable hydrogel containing adipose derived stem cells to promote vascularization for skin wounds and tissue engineering. Biomaterials 2017, 129, 188−198. (18) Kobsa, S.; Kristofik, N. J.; Sawyer, A. J.; Bothwell, A. L. M.; Kyriakides, T. R.; Saltzman, W. M. An electrospun scaffold integrating nucleic acid delivery for treatment of full thickness wounds. Biomaterials 2013, 34, 3891−3901. (19) Zhang, W.; Chen, L.; Chen, J.; Wang, L.; Gui, X.; Ran, J.; Xu, G.; Zhao, H.; Zeng, M.; Ji, J.; Qian, L.; Zhou, J.; Ouyang, H.; Zou, X. Silk fibroin biomaterial shows safe and effective wound healing in animal models and a randomized controlled clinical trial. Adv. Healthcare Mater. 2017, 6 (10), 1700121. (20) Dong, R. H.; Jia, Y. X.; Qin, C. C.; Zhan, L.; Yan, X.; Cui, L.; Zhou, Y.; Jiang, X.; Long, Y. Z. In situ deposition of a personalized nanofibrous dressing via a handy electrospinning device for skin wound care. Nanoscale 2016, 8, 3482−3488. (21) Yang, X.; Yang, J.; Wang, L.; Ran, B.; Jia, Y.; Zhang, L.; Yang, G.; Shao, H.; Jiang, X. Pharmaceutical intermediate-modified gold nanoparticles: against multidrug-resistant bacteria and wound-healing application via an electrospun scaffold. ACS Nano 2017, 11, 5737− 5745. (22) Zhang, H.; Xia, J. Y.; Pang, X. L.; Zhao, M.; Wang, B. Q.; Yang, L. L.; Wan, H. S.; Wu, J. B.; Fu, S. Z. Magnetic nanoparticle-loaded electrospun polymeric nanofibers for tissue engineering. Mater. Sci. Eng., C 2017, 73, 537−543. (23) Li, W. P.; Su, C. H.; Wang, S. J.; Tsai, F. J.; Chang, C. T.; Liao, M. C.; Yu, C. C.; Vi Tran, T. T.; Lee, C. N.; Chiu, W. T.; Wong, T. W.; Yeh, C. S. CO2 delivery to accelerate incisional wound healing following single irradiation of near-infrared lamp on the coordinated colloids. ACS Nano 2017, 11, 5826−5835. (24) Bellas, E.; Lo, T. J.; Fournier, E. P.; Brown, J. E.; Abbott, R. D.; Gil, E. S.; Marra, K. G.; Rubin, J. P.; Leisk, G. G.; Kaplan, D. L. Injectable silk foams for soft tissue regeneration. Adv. Healthcare Mater. 2015, 4 (3), 452−459. (25) Bankoti, K.; Rameshbabu, A. P.; Datta, S.; Maity, P. P.; Goswami, P.; Datta, P.; Ghosh, S. K.; Mitra, A.; Dhara, S. Accelerated

CONCLUSIONS In summary, we have developed an injectable hybrid alginate hydrogel cross-linked by calcium gluconate deposited in PCEC porous microspheres. Fibroblasts (3T3 and L929) can efficiently attach and stretch on both porous microspheres and MPs/Alg hydrogel. In the marine full-thickness skin defect modal, MPs/Alg hydrogel increased the wound healing rate in the early regeneration period and promoted complete skin auxiliary in the final healing process by gross assessment, quantitative measurement, and histological evaluation throughout the wound healing. This work demonstrated that MPs/Alg hybrid hydrogel enhanced the cutaneous wound healing process. What’s more, the MPs/Alg hydrogel was simple to produce and easy to handle, which may have great potential as a skin dressing for further clinical application.

■

AUTHOR INFORMATION

Corresponding Author

*Tel./Fax: +86-28-85501986. E-mail: [email protected]. ORCID

ZhiYong Qian: 0000-0003-2992-6424 Author Contributions #

The authors have contributed equally to this work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was financially supported by The National Key Research and Development Program of China (2017YFC1103500, 2017YFC1103502), the National Natural Science Foundation (31525009), Sichuan Innovative Research Team Program for Young Scientists (2016TD0004), and Distinguished Young Scholars of Sichuan University (2011SCU04B18).

■

REFERENCES

(1) Gurtner, G. C.; Werner, S.; Barrandon, Y.; Longaker, M. T. Wound repair and regeneration. Nature 2008, 453, 314−321. (2) Li, Z. W.; Li, C. W.; Wang, Q.; Shi, S. J.; Hu, M.; Zhang, Q.; Cui, H. H.; Sun, J. B.; Zhou, M.; Wu, G. L.; Dang, J. Z.; Lu, L. C. The cellular and molecular mechanisms underlying silver nanoparticle/ chitosan oligosaccharide/poly(vinyl alcohol) nanofiber-mediated wound healing. J. Biomed. Nanotechnol. 2017, 13, 17−34. (3) Lee, J. K.; Kang, S. M.; Yang, S. H.; Cho, W. K. Micro/ Nanostrctured films and adhesives for biomedical applications. J. Biomed. Nanotechnol. 2015, 11, 2081−2110. (4) Rnjak-Kovacina, J.; Wise, S. G.; Li, Z.; Maitz, P. K. M.; Young, C. J.; Wang, Y.; Weiss, A. S. Electrospun synthetic human elastin: collagen composite scaffolds for dermal tissue engineering. Acta Biomater. 2012, 8, 3714−3722. (5) Levengood, S. L.; Erickson, A. E.; Chang, F.; Zhang, M. Chitosanpoly(caprolactone) nanofibers for skin repair. J. Mater. Chem. B 2017, 5 (9), 1822−1833. (6) Chen, L.; Xing, Q.; Zhai, Q.; Tahtinen, M.; Zhou, F.; Chen, L.; Xu, Y.; Qi, S.; Zhao, F. Pre-vascularization enhances therapeutic effects of human mesenchymal stem cell sheets in full thickness skin wound repair. Theranostics 2017, 7 (1), 117−131. (7) Lombardi, B.; Casale, C.; Imparato, G.; Urciuolo, F.; Netti, P. A. Spatiotemporal evolution of the wound repairing process in a 3D human dermis equivalent. Adv. Healthcare Mater. 2017, 6 (13), 1601422. (8) Kubinova, S.; Zaviskova, K.; Uherkova, L.; Zablotskii, V.; Churpita, O.; Lunov, O.; Dejneka, A. Non-thermal air plasma G

DOI: 10.1021/acsbiomaterials.7b00860 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Article

ACS Biomaterials Science & Engineering healing of full thickness dermal wounds by macroporous waterborne polyurethane-chitosan hydrogel scaffolds. Mater. Sci. Eng., C 2017, 81, 133−143. (26) Gong, C. Y.; Wu, Q. J.; Wang, Y. J.; Zhang, D. D.; Luo, F.; Zhao, X.; Wei, Y. Q.; Qian, Z. Y. A biodegradable hydrogel system containing curcumin encapsulated in micelles for cutaneous wound healing. Biomaterials 2013, 34, 6377−6387. (27) Li, B.; Wang, L.; Xu, F.; Gang, X.; Demirci, U.; Wei, D.; Li, Y.; Feng, Y.; Jia, D.; Zhou, Y. Hydrosoluble, UV-crosslinkable and injectable chitosan for patterned cell-laden microgel and rapid transdermal curing hydrogel in vivo. Acta Biomater. 2015, 22, 59−69. (28) Wang, R.; Li, Q.; Chi, B.; Wang, X.; Xu, Z.; Xu, Z.; Chen, S.; Xu, H. Enzyme-induced dual-network ε-poly-L-lysine-based hydrogels with robust self-healing and antibacterial performance. Chem. Commun. 2017, 53, 4803−4806. (29) Liu, C.; Gong, C.; Huang, M.; Wang, J.; Pan, Y.; Zhang, Y.; Li, G.; Gou, M.; Wang, K.; Tu, M.; Wei, Y.; Qian, Z. Thermoreversible gel-sol behavior of biodegradable PCL-PEG-PCL triblock copolymer in aqueous solutions. J. Biomed. Mater. Res., Part B 2008, 84, 165−175. (30) Ji, Y.; Ghosh, K.; Shu, X. Z.; Li, B.; Sokolov, J. C.; Prestwich, G. D.; Clark, R. A. F.; Rafailovich, M. H. Electrospun three-dimensional hyaluronic acid nanofibrous scaffolds. Biomaterials 2006, 27, 3782− 3792. (31) Gou, M. L.; Zheng, L.; Peng, X. Y.; Men, K.; Zheng, X. L.; Zeng, S.; Guo, G.; Luo, F.; Zhao, X.; Chen, L. J.; Wei, Y. Q.; Qian, Z. Y. Poly(ε-caprolactone)-poly(ethylene glycol)-poly(-caprolactone) (PCL-PEG-PCL) nanoparticles for honokiol delivery in vitro. Int. J. Pharm. 2009, 375, 170−176. (32) Dai, M.; Xu, X.; Song, J.; Fu, S. Z.; Gou, M. L.; Luo, F.; Qian, Z. Y. Preparation of camptothecin-loaded PCEC microspheres for the treatment of colorectal peritoneal carcinomatosis and tumor growth in mice. Cancer Lett. 2011, 312, 189−196. (33) Liao, J. F.; Li, W. T.; Peng, J. R.; Yang, Q.; Li, H.; Wei, Y. Q.; Zhang, X. N.; Qian, Z. Y. Cominbined cancer photothermalchemotherapy based on doxorubicin/gold nanorod-loaded polymersomes. Theranostics 2015, 5 (4), 345−356. (34) Liao, J. F.; Wang, B. Y.; Huang, Y. X.; Qu, Y.; Peng, J. R.; Qian, Z. Y. Injectable alginate hydrogel cross-linked by calcium gluconateloaded porous microspheres for cartilage tissue engineering. ACS Omega 2017, 2, 443−454. (35) Liu, X.; Jin, X.; Ma, P. X. Nanofibrous hollow microspheres selfassembled from star-shaped polymers as injectable cell carriers for knee repair. Nat. Mater. 2011, 10, 398−406. (36) Martin, P. Wound healing-aiming for perfect skin regeneration. Science 1997, 276, 75−81. (37) Zhao, X.; Wu, H.; Guo, B.; Dong, R.; Qiu, Y.; Ma, P. X. Antibacterial anti-oxidant electroactive injectable hydrogel as selfhealing wound dressing with hemostasis and adhesiveness for cutaneous wound healing. Biomaterials 2017, 122, 34−47.

H

DOI: 10.1021/acsbiomaterials.7b00860 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX