Combined chemical and structural signals of biomaterials synergistically activate cell-cell communications for improving tissue regeneration

Acta Biomaterialia xxx (2017) xxx–xxx

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Combined chemical and structural signals of biomaterials synergistically activate cell-cell communications for improving tissue regeneration Yachen Xu a,c, Jinliang Peng b, Xin Dong a,c, Yuhong Xu b, Haiyan Li a,c,⇑, Jiang Chang c,d,⇑ a

Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, School of Biomedical Engineering, Shanghai Jiao Tong University, 1954 Huashan Road, Shanghai 200030, China School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China c Med-X Research Institute, School of Biomedical Engineering, Shanghai Jiao Tong University, 1954 Huashan Road, Shanghai 200030, China d Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Dingxi Road, Shanghai 200050, China b

a r t i c l e

i n f o

Article history: Received 21 November 2016 Received in revised form 24 February 2017 Accepted 31 March 2017 Available online xxxx Keywords: Tissue engineering Chemical signals Structural signals Co-cultures Skin tissue regeneration

a b s t r a c t Biomaterials are only used as carriers of cells in the conventional tissue engineering. Considering the multi-cell environment and active cell-biomaterial interactions in tissue regeneration process, in this study, structural signals of aligned electrospun nanofibers and chemical signals of bioglass (BG) ionic products in cell culture medium are simultaneously applied to activate fibroblast-endothelial cocultured cells in order to obtain an improved skin tissue engineering construct. Results demonstrate that the combined biomaterial signals synergistically activate fibroblast-endothelial co-culture skin tissue engineering constructs through promotion of paracrine effects and stimulation of gap junctional communication between cells, which results in enhanced vascularization and extracellular matrix protein synthesis in the constructs. Structural signals of aligned electrospun nanofibers play an important role in stimulating both of paracrine and gap junctional communication while chemical signals of BG ionic products mainly enhance paracrine effects. In vivo experiments reveal that the activated skin tissue engineering constructs significantly enhance wound healing as compared to control. This study indicates the advantages of synergistic effects between different bioactive signals of biomaterials can be taken to activate communication between different types of cells for obtaining tissue engineering constructs with improved functions. Statement of Significance Tissue engineering can regenerate or replace tissue or organs through combining cells, biomaterials and growth factors. Normally, for repairing a specific tissue, only one type of cells, one kind of biomaterials, and specific growth factors are used to support cell growth. In this study, we proposed a novel tissue engineering approach by simply using co-cultured cells and combined biomaterial signals. Using a skin tissue engineering model, we successfully proved that the combined biomaterial signals such as surface nanostructures and bioactive ions could synergistically stimulate the cell-cell communication in coculture system through paracrine effects and gap junction activation, and regulated expression of growth factors and extracellular matrix proteins, resulting in an activated tissue engineering constructs that significantly enhanced skin regeneration. Ó 2017 Published by Elsevier Ltd on behalf of Acta Materialia Inc.

1. Introduction At the beginning of tissue engineering technology, only one type of cells was used to combine with biomaterials in order to

⇑ Corresponding authors at: Med-X Research Institute, School of Biomedical Engineering, Shanghai Jiao Tong University, 1954 Huashan Road, Shanghai 200030, China. E-mail address: [email protected] (J. Chang).

reconstruct a specific tissue [1,2]. However, it is known that multiple cell types are often involved in most of tissue regeneration processes, and previous studies have found that cell-cell interactions may significantly affect the tissue regeneration process [3,4]. In addition, another limitation of the conventional tissue engineering approach is that biomaterial scaffolds are only considered as the physical carrier of cells and growth factors, but the potential bioactive effects of biomaterials are ignored. In recent years, the role of biomaterials in tissue engineering has been re-considered, and

http://dx.doi.org/10.1016/j.actbio.2017.03.056 1742-7061/Ó 2017 Published by Elsevier Ltd on behalf of Acta Materialia Inc.

Please cite this article in press as: Y. Xu et al., Combined chemical and structural signals of biomaterials synergistically activate cell-cell communications for improving tissue regeneration, Acta Biomater. (2017), http://dx.doi.org/10.1016/j.actbio.2017.03.056

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more and more studies have been focusing on the bioactivity of biomaterials to stimulate cell differentiation, growth factor expression and tissue regeneration [5–7]. In particular, some recent studies have demonstrated that both chemical signals such as ions released from biomaterials and structural signals such as surface micro/nano structures have the abilities to stimulate stem cell differentiation and tissue regeneration [8–11]. Among these studies, chemical signals of bioglass (BG), mainly ionic products, have been widely reported to be able to promote matrix synthesis of fibroblasts and angiogenic differentiation of endothelial cells (ECs) [12,13]. In addition, a number of studies have demonstrated that electrospun nanofibers prepared by a mixture of biocompatible polymers, poly (D, L-lactide) (PDLLA) and polycaprolactone (PCL) have shown excellent mechanical strength and stability in cell culture medium [14,15]. Furthermore, the morphology and alignment of PDLLA/PCL electrospun nanofibers, especially aligned arrangement of nanofibers, have profound effects on morphology and behaviors of ECs and fibroblasts, which can significantly stimulate angiogenesis and tissue regeneration [16,17]. However, most studies only investigated the effects of single type of biomaterial signals on behaviors of single type of cells. Although much attention has been paid on cell-cell interaction for tissue engineering, and our previous studies reported the effects of biomaterials as cell carriers on cell-cell interactions in tissue engineering [18,19], the specific roles of different biomaterial signals and the interactions between different biomaterial signals on cell-cell communications during the tissue engineering process has rarely been seriously considered. As angiogenesis is critical for wound healing and interactions between ECs and fibroblasts played important roles in wound healing, in this study, we chose PDLLA/PCL electrospun nanofibers combined with BG to investigate the combinatory effects of the structural and chemical signals on cell-cell interactions of ECs and fibroblasts. Recent years, significant progress has been made in the development of skin tissue engineering, and the tissue-engineered skin substitutes range from non-cellular polymer scaffolds, such as collagen/chitosan porous scaffolds [20], electrospun poly(lactic acidco-glycolic acid) scaffolds [21] to non-cellular biological scaffolds, such as small intestinal submucosa [22], extracellular matrix [23] and cellular devices [24]. As for the cellular skin substitutes, cells, including fibroblasts [25], epidermal cells [26], dermal cells [27] and stem cells [28], have been either applied with biodegradable scaffolds or applied as cell sheets without substrates [29]. Although electrospun mats with random nanofibers have been applied as substrates for skin tissue engineering, the effects of electrospun mats with aligned nanofibers have seldom been used for skin regeneration. In addition, the previous studies of skin tissue engineering constructs have rarely discussed the effects of bioactive biomaterials on cell-cell interactions between critical cells involved in wound healing. Furthermore, combination effects of different biomaterial stimulatory effects on cell behaviors or cellcell interactions involved in wound healing have never been studied, which is important for designing bioactive tissue engineering scaffolds to enhance skin regeneration. Therefore, in the present study, we proposed a novel skin tissue engineering construct by activating co-cultured cells using combination of two different biomaterial signals. Our hypothesis is that the co-cultured cell system activated by combination of chemical signals such as bioactive ions and structural signals such as aligned nano-fibrous structure may significantly enhance tissue regeneration as compared with traditional tissue engineering approach. To prove the concept, a co-culture of human dermal fibroblasts (HDFs) and human umbilical vein endothelial cells (HUVECs) was used as the co-culture cell model, and aligned electrospun nanofibers and bioglass ionic products added in cell culture medium were used as structural and chemical stimulatory signals of bioma-

terials for activating co-cultured cells, respectively. We first proved the effects of combined biomaterial signals on the communications between co-cultured HDFs and HUVECs and analyzed the specific role of each type of signals to elucidate the mechanisms of the activation. Then, we applied HDFs-HUVECs co-cultured skin tissue engineering construct activated by combined stimulatory signals of biomaterials to full-thickness excisions on mouse back and investigated the angiogenesis and protein synthesis in wound area. Our results demonstrated that tissue engineering can be significantly improved through co-cultured cell systems activated by biomaterials, and the design of bioactive materials to optimize synergistic bioactive signals is an effective way to obtain activated tissue engineering constructs for tissue regeneration and wound healing applications. 2. Materials and methods 2.1. Electrospun nanofibrous scaffolds Poly (D, L-lactide) (PDLLA, Mw = 45 kDa) was purchased from Jinan Daigang Biomaterial Co, Ltd. (Shandong, China). Polycaprolactone (PCL, Mw = 80 kDa) was purchased from Sigma Co. N,Ndimethyl formamide (DMF) and tetrahydrofuran (THF) were purchased from Sinopharm Chemical Reagent Co. (Shanghai, China). Electrospun nanofibers were prepared according to the literatures [15,30]. Briefly, the blend of PDLLA and PCL with a certain mass ratio (w/w = 50/50) was dissolved in a mixture of DMF and THF (v/v = 4/1), and then stirred for 6 h to obtain a homogeneous and stable solution with polymer concentration of 4.8% (w/v). The flow rate of the solution in the syringe (2 mL) was 0.02 mL/m 1 by using a syringe pump (LSP01-1A, Baoding Longer Precision Pump, China). A high voltage power supply (Dongwen, China) was used, and the voltage applied to the needle of the syringe was 8 kV. The distance between the tip of the needle and the collector was 15 cm, and the collecting time was fixed for 1 h. To prepare electrospun scaffolds with random and aligned nanofibers, aluminum foil and highspeed roller (rotating speed = 2000 rpm) were used as collectors, respectively [15,30]. All the experiments were conducted at room temperature and the relative humidity was about 40–60%. All the electrospun scaffolds were vacuum dried for 24 h to completely remove any residual solvent. Morphology and microstructure of the electrospun scaffolds were observed using a scanning electron microscope (SEM) (S4800, Hitachi, Japan). Briefly, the scaffolds were cut into 5 mm  5 mm and attached to the sample stage by conductive adhesive, after gold-plating, the samples were observed and photographed using SEM. Surface wettability of the scaffolds were evaluated by measuring the static water contacting angles (WCA) using a Kruss GmbH DSA 100 Mk 2 goniometer (Hamburg, Germany), followed by image processing of sessile drops using a DataPhysics OCA20 CA system. Water droplets in 3.0 lL were dropped onto the surfaces of the electrospun scaffolds, and the average WCA value was obtained by measuring the water droplets set at 5 randomly distributed positions. Electrospun scaffolds were cut into squares with dimensions of 10 mm  10 mm and 25  25 mm for 24-well plates and 6-well plates, respectively. The obtained scaffold squares were sterilized after being soaked in 75% alcohol for 20 min for further cell culture. 2.2. BG ion extracts BG powders (over 200 mesh) were kindly provided by Shanghai Institute of Ceramics, Chinese Academy of Science. BG ion extracts were prepared according to the methods reported in literatures adapted from ISO10993-1 procedures [31,32]. Briefly, 1 g of BG

Please cite this article in press as: Y. Xu et al., Combined chemical and structural signals of biomaterials synergistically activate cell-cell communications for improving tissue regeneration, Acta Biomater. (2017), http://dx.doi.org/10.1016/j.actbio.2017.03.056

Y. Xu et al. / Acta Biomaterialia xxx (2017) xxx–xxx

powders was soaked in 5 mL of serum-free endothelial culture medium (ECM) (Sciencell, USA) and Dulbecco modified Eagle medium (DMEM) (GIBCO, USA), respectively. The mixture was incubated for 24 h in a humidified 37 °C/5% CO2 incubator. The supernatant was then collected and sterilized through a filter (Millipore, 0.22 lm). For further use, BG ion extracts were diluted with total ECM (endothelial cell basal medium + 5% fetal bovine serum (FBS) + 1% endothelial cell growth supplement + 1% penicillinstreptomycin (P/S)) and total DMEM (DMEM + 10% FBS + 1% P/S) at ratios of 1/128, respectively. The medium containing BG ion extracts with dilution of 1/128 were recorded as 1/128BG, while the control medium without BG ion extracts were recorded as 0BG. The concentrations of Ca, Si, and P in the diluted ion extracts were detected by inductively coupled plasma atomic emission spectroscopy (ICP-AES) (Optima 3000DV, PerkinElmer, USA). Concentrations of ions in different culture media were recorded in Table S1. 2.3. Cell isolation and culture HUVECs were isolated from human umbilical cord veins according to the method reported previously [33] and HDFs were isolated from the superficial layer of adult human skin dermatome according to previous work [34]. An Institutional Review Committee of Shanghai Jiao Tong University, School of Biomedical Engineering approved all these protocols. Endothelial cell basal medium with 5% FBS, 1% endothelial cell growth supplement and 1% P/S (total ECM) was used as HUVECs’ culture medium, while DMEM with 10% FBS and 1% P/S (total DMEM) was used as HDFs’ culture medium. The culture medium was replaced every 3 days, and only early passages (passages 2–7) of the HUVECs and HDFs were used in this study. 2.4. Effects of combined biomaterial signals on morphology of HUVECs and HDFs The BG ion extracts diluted at 1/128 and electrospun scaffolds with random and aligned nanofibers were used to determine the effects of chemical signals of BG ionic products and structural signals of nanofibers on morphology of HUVECs and HDFs. Total ECM and total DMEM without BG ion extracts were used as control medium for HUVECs and HDFs, respectively. Cells seeded on coverslips were regarded as non-structure control groups. According to our previous studies [18], 3 days of culture is long enough for vascularization of endothelial cells in the HDF-HUVEC co-cultures and for observing the effects of biomaterials on vascularization of endothelial cells in the HDF-HUVEC co-cultures. Therefore, we chose day 3 as the time point for characterization cells. To investigate cell morphology and distribution on the electrospun scaffolds with different structures, F-actin staining was applied on HUVECs and HDFs after cells were cultured for 3 days. Rodamine phalloidin R415 (Invitrogen, USA) and 4–6-diamidino2-phenylindole (DAPI) (FluoProbes, USA) were applied to stain actin filaments and nuclei of HUVECs and HDFs according to the supplier’s procedure, and the cytoskeletal and nuclear organizations were observed and photographed using a confocal microscope (Leica TCS SP5, Germany) equipped with a CCD camera (Leica DFC 420C, Germany). 2.5. Effects of combined biomaterial signals on HDF-HUVEC cocultures The HDF-HUVEC direct contact co-culture model was established according to procedures reported in a literature [18]. HDFs were first seeded on coverslips and electrospun scaffolds with different structures at a density of 8  104 cells per well in 24-well

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plates and 2  105 cells per well in 6-well plates, respectively. After HDFs were cultured with control medium for 12 h, HUVECs were seeded on HDFs at a density of 1.2  105 cells per well in 24-well plates and 3  105 cells per well in 6-well plates, respectively. At the same time, the culture medium was replaced by BG ion extracts diluted with mixed total DMEM and total ECM (v/ v = 1/1) at 1/128. The mixed medium of total DMEM and total ECM (v/v = 1/1) was considered as control medium. After being co-cultured for 3 days, the cells in 24-well plates were fixed with 4% (w/v) paraformaldehyde (PFA) (Dingguo Chemical Reagent Co., Shanghai, China) for immunofluorescence staining. 2.6. Quantitative real-time polymerase chain reaction (Q-RT- PCR) At the determined time points, mono-cultured or co-cultured cells in 6-well plates were washed twice with cold phosphate buffered saline (PBS) and HUVECs were separated from HDFs in cocultures. Briefly, co-cultured cells in 6-well plates were collected by trypsinization. To separate HUVECs from HDFs, the cocultured cells were mixed with magnetic beads coupled with an antibody against CD31 (Invitrogen, USA) in 1.5 ml Eppendorf tubes, and shook for 30 min in 4 °C. Then, the tubes were fixed on the Magnetic Separation Rack (Invitrogen, USA) for 5 min. During this process, the endothelial cells conjugated with magnetic beads aggregated to the inner side of the tube that is close to the magnet. The supernatants were separated from the tubes for collecting cocultured HDFs, and the magnetic beads attached to the inner side of the tube that is close to the magnet were co-cultured HUVECs. The procedure was according to the method established by Guillotin et al. [35]. The separated HUVECs and HDFs were named co-HUVEC and co-HDF, respectively. RNA was extracted using E. Z. N. A Total RNA kit I (OMEGA, Biotek, USA) according to the instructions. The concentration of RNA was measured by a Nanodrop 1000 reader (Thermo Scientific, USA) and cDNA was synthesized using a ReverTra Ace-a kit (Toyobo, Japan) according to the instructions. cDNA was diluted at 1:20 with sterilized deionized water. Then, 4.2 ll of diluted cDNA was mixed with 5.8 ll of SYBR-Green (ToYOBO, Japan) and primers (Sangon Biotech, China). Primers of vascular endothelial growth factor (VEGF), VEGF receptor 2 (KDR), endothelial nitric oxide synthase (eNOS), collagen I, elastin, fibronectin, connexin 43 (Cx43) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used at a final concentration of 400 nM. GAPDH was used as a housekeeping gene. The sequences for primers are listed in Table S2. The mixture was loaded in a 384-well plate and analyzed by 7900 Real-time PCR system (Applied Biosystems, USA), which performed 40 cycles (95 °C for 15 s, 60 °C for 15 s, 72 °C for 45 s) followed by a 1 min denaturation at 95 °C. Each reaction was performed in triplicate, and data were analyzed by DDCt method. The data were then normalized to GAPDH gene expression of each condition and compared to the corresponding gene expression in control samples. 2.7. Immunofluorescence staining Immunofluorescence staining of von Willebrand factor (vWF) was applied on co-cultures to observe the distribution of HUVECs and tubule formation. Immunofluorescence staining of collagen I, elastin and fibronectin were applied on co-cultures to detect the location and expression of collagen I, elastin and fibronectin. Immunofluorescence staining of Cx43 was applied on co-cultures to explore its location and expression. After being co-cultured on electrospun scaffolds with different structures and coverslips in 24-well plates for 3 days, the cells were washed twice with PBS and fixed with 4% PFA at room temperature for 15 min. Then, the cells were permeabilized with

Please cite this article in press as: Y. Xu et al., Combined chemical and structural signals of biomaterials synergistically activate cell-cell communications for improving tissue regeneration, Acta Biomater. (2017), http://dx.doi.org/10.1016/j.actbio.2017.03.056

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methanol for 5 min and blocked with PBS containing 1% (w /v) bovine serum albumin (BSA) for 1 h at 37 °C. After blocking and washing, primary antibody solution containing rabbit anti-vWF, rabbit anti-collagen I, mouse anti-elastin, rabbit anti-fibronectin or rabbit anti-Cx43 (all purchased from Abcam, USA and diluted in PBS-0.5% BSA at 1:300) was added to the cells and incubated at 37 °C for 2 h. The cells were then incubated overnight at 4 °C, and washed twice with PBS. Alexa 488 goat anti-rabbit IgG (Invitrogen, USA) or Alexa 488 goat anti-mouse IgG (Invitrogen, USA) secondary antibody (diluted in PBS-0.5% BSA at 1/1000) was used to incubate the cells at 37 °C for 1 h. Finally, nuclei were revealed by incubating the cells with 1 lg/ml DAPI for 10 min at room temperature. At the end, the electrospun scaffolds or coverslips with cells were removed from plate wells and mounted on slides. The stained cells were observed with a confocal microscope (Leica TCS SP5, Germany) and images were taken by a CCD camera (Leica DFC 420C, Germany). 2.8. Preparation of skin tissue engineering constructs According to the in vitro results, aligned nanofibers with cocultures of HDFs and HUVECs were further applied for skin tissue engineering in vivo. The HDF-HUVEC co-cultures can form a cell sheet on culture substrates as long as the culture time is long enough, which can be used as skin tissue engineering constructs. In this study, HDF-HUVEC co-cultures cultured on electrospun scaffolds with aligned nanofibers and with or without BG ion extracts diluted at 1/128 ratio were used to obtain electrospun 1/128BG and 0BG co-culture constructs, respectively. To obtain a co-culture construct without being affected by structural signals, HDF-HUVEC co-cultures cultured on a thermos-responsive culture dish without any special structure and with or without BG ion extracts diluted at 1/128 ratio were used to obtain 1/128BG and 0BG co-culture constructs, respectively. The thermos-responsive culture dish has a controllable surface [35] the surface is hydrophobic at 37 °C but hydrophilic at the temperature below 32 °C. As a result, when the cells seeded on the dish are cultured for a certain period, a co-culture construct will form and this coculture construct can be detached from the surface of culture dish by changing the temperature, and immediately transferred to a polyvinylidene fluoride (PVDF) membranes provided by the thermo-responsive culture dish supplier according to the instructions of supplier. The PVDF film is chemically inert and smooth, which has little chemical or structural effects on cell sheet. Briefly, HDFs were directly seeded on electrospun scaffolds with aligned nanofibers in 6-well plates or Upcell 3.5 cm thermosresponsive dishes (Nunc, Thermo Scientific, USA) at a density of 2  105 cells per well/dish. After 12 h, HUVECs were seeded on HDFs at the density of 3  105 cells per well/dish and the culture medium were replaced by BG ion extracts diluted with mixed total DMED and total ECM (v/v = 1/1) at 1/128 (1/128BG) or pure mixed medium of total DMEM and total ECM (v/v = 1/1) without BG ion extracts (0BG). After being co-cultured for 3 days, co-cultures formed on aligned electrospun scaffolds were directly collected by removing the electrospun scaffolds from wells and were named as electrospun 1/128BG and 0BG co-culture construct, respectively. The constructs were cut into four round pieces with 1 cm diameter for further animal studies. The co-cultures formed on thermosresponsive culture dishes were obtained according to the manufacturer’s instructions and were named as 1/128BG and 0BG coculture construct, respectively. At the end of culture, a polymembrane was placed over the cells and incubated at room temperature (25 °C) for 10 min for the construct detachment. After being detached, the co-culture constructs attaching with the membrane were collected and cut into four pieces with 1 cm diameter for implantation in animal studies.

2.9. Application of skin tissue engineering constructs in full-thickness excisional wound models An Institutional Review Committee of Shanghai Jiao Tong University, School of Biomedical Engineering approved all animal study protocols. As recombinant human epidermal growth factor has been widely reported to be able to accelerate wound healing and it has been commercially applied in clinics [36,37], in this study, we used the wounds treated with recombinant human epidermal growth factor gel as positive control. For animal study, 3 time points (2, 7, and 14 days) were set. For each time point in one individual experiment, the number of full-thickness excisions is 6 as there were 6 experiment groups. Therefore, for 3 time points and 3 individual experiments, the total number of full-thickness excisions is 6  3  3 = 54. Since two full-thickness excisions were made on one mouse back, 27 nude mice (BALB/c, 6–8 weeks) were needed for animal studies. The mice were purchased from Shanghai Laboratory Animal Center. After that, the prepared co-culture constructs, including electrospun 0BG and 1/128BG co-culture constructs as well as 0BG and 1/128BG co-culture constructs, were placed on the wound site. The wounds left untreated were recorded as negative control, while the wounds treated with recombinant human epidermal growth factor gel (Pavay Gene pharmaceutical, China) were recorded as positive control. After all constructs were applied on the wounds, all wounds were covered with a Tegaderm Film (1626 W, 3 M, USA) and bandages to protect the treatment. Finally, in order to delay the healing of wounds, Depo-Medrol (20 mg/kg BW) (Pfizer) was injected intramuscularly to all mice at day 0. 2.10. Analysis of wound healing 2.10.1. Wound closure At each determined time point, the wound size was measured and the mice were euthanized by anesthetic injection. The percentage of wound closure was calculated as: % of wound closure = (area of original wound area of actual wound)/(area of original wound)  100, n = 3. 2.10.2. Histological analysis and immunohistochemistry staining At each time point, the tissue samples were collected and fixed in 4% PFA over 24 h. After being dehydrated and transparentized, the tissues were embedded in paraffin and cross-sectioned longitudinally into 5 lm sections using a Leica RM2245 microtome for further histological staining and analysis. For histological analysis, the tissue sections collected on glass slides were rehydrated and stained with hematoxylin and eosin (HE) to visualize the overall tissue morphology. For immunohistochemistry staining, rabbit anti-CD31 (Abcam, USA) and rabbit anti-collagen I (Abcam, USA) were used to detect blood vessel formation and extracellular matrix synthesis, respectively. Briefly, the tissue sections were rehydrated and immersed in 0.01 M sodium citrate (Sinopharm Chemical Reagent, China) at 99 °C for 20 min to improve antigen exposure. After cooling down to room temperature, the sections were immersed in 0.3% H2O2/methanol (v/v) to terminate peroxidase activity. Subsequently, all sections were blocked with 5% BSA in PBS for 1 h at room temperature, and then incubated in a primary antibody solution diluted with 1% BSA in PBS with CD31 antibody (diluted at 1:50) and collagen I antibody (diluted at 1:300) overnight at 4 °C. Next, the sections were rinsed with PBS and incubated with a second antibody solution in DAB kit (Gene Tech, Shanghai). Finally, the slides were counterstained with hematoxylin, dehydrated and mounted. An upright microscope (Leica DM2500, Germany) was used to observe the stained sections and a CCD camera (Leica DFC 420C, Germany) was used to take images. Blood vessels were identified in CD31 stained tissues at

Please cite this article in press as: Y. Xu et al., Combined chemical and structural signals of biomaterials synergistically activate cell-cell communications for improving tissue regeneration, Acta Biomater. (2017), http://dx.doi.org/10.1016/j.actbio.2017.03.056

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200 magnification by defining lumens, identifying the presence of red blood cells within their boundaries and locating brown lines that showed up with CD31 positive labeling. Vessel density was the number of vessels in a field in the upright microscope with a magnification of 200. In each sample, three fields were randomly chosen for taking images, and three samples were taken images for each condition. 2.11. Statistical analysis Data were expressed as means ± standard deviation. Three independent experiments were carried out for validity, and at least three samples per test were taken for statistical analysis. Statistical significance between groups was calculated using two-tailed analysis of variance (ANOVA) and performed with a Student’s t test program. The differences were considered significant when p < 0.05 (⁄, #,% or D) or p < 0.01 (⁄⁄, ##,%% or DD). 3. Results 3.1. Characterization of electrospun nanofibers and BG ion extracts SEM and contact angle analysis of electrospun nanofibers have been shown in Fig. S1. Fig. S1a shows SEM images of random and aligned electrospun nanofibers. It can be seen that the nanofibers are uniform in diameter. Fig. S1b–c show that all scaffolds are hydrophobic. The electrospun scaffolds with aligned nanofibers have a water contact angle similar to the electrospun scaffolds with random nanofibers. The ion concentrations of BG ion extracts diluted at 1/128 with different control media are shown in Table S1. There is little difference between the concentrations of Ca and P ion in BG containing medium and corresponding control medium. However, the concentrations of Si ions were significantly increased when BG ion extracts were diluted with control medium. The concentration of Si ions in ECM containing 1/128 BG and DMEM containing 1/128BG is about 1.14 mg/ml and 0.88 mg/ml, respectively. 3.2. Effects of combined biomaterial signals on vascular growth factor secretion and vascularization in HDF-HUVEC co-culture Combining biomaterial signals such as structural signals of electrospun nanofibers and chemical signals of BG to activate cell-cell interactions is the key of our novel approach. To confirm the concept, HDF and HUVEC were co-cultured in a direct contact model under different conditions. One critical question is whether the combined biomaterial signals can regulate growth factor expression of co-cultured cells in tissue engineering constructs. Considering the importance of angiogenesis in wound healing process, the effects of chemical signals of BG ionic products and structure signals of electrospun nanofibers on VEGF, KDR and downstream angiogenic molecule eNOS in co-cultured cells were evaluated (Fig. 1a–c). It is interesting to see that, as compared to control group, aligned nanofibers with medium containing 1/128BG resulted in a 10 time-increase of the expression of VEGF in coHDFs. In contrast, when cells were cultured with different structural signals but same chemical signals, as compared to their corresponding control groups, electrospun scaffolds with aligned nanofibers resulted in about 2.5 times increase of the VEGF expression in co-HDF, while random nanofibers upregulated the VEGF expression for about 1.2–1.5 times. Meanwhile, when cells were cultured with same structural signals, the chemical signal of 1/128BG stimulated the expression of VEGF in co-HDF for about 2.5 times (Fig. 1a). These results indicate that the expression of VEGF stimulated by combined biomaterial signals is significantly

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higher than that of VEGF stimulated by the simple stimulation of the single signal, and suggests a synergetic effect of the combined signals. Another interesting new finding is that the typical paracrine effects of stimulation on VEGF expression by both single structural signal as well as the combined chemical and structural signals of biomaterials were observed in the co-cultures. The evidence is that the co-HDF expressed high level of VEGF, while co-HUVECs expressed high level of VEGF’s receptor KDR (Fig. 1a–b). Interestingly, as compared to control group, aligned nanofibers with medium containing 1/128BG resulted in 5.5 times increase for KDR and 30 times increase for eNOS expression in co-HUVEC, respectively, which were much higher than those with the simple addition of single signal stimulation (Fig. 1b–c). Furthermore, when cells were cultured with different structural signals but same chemical signals, as compared to control group, electrospun scaffolds with aligned nanofibers significantly increased the expression of KDR and eNOS in co-HUVEC for about 2.5 times. Meanwhile, when cells were cultured with same structural signals, 1/128BG significantly upregulated the expression of KDR and eNOS in co-HUVEC for about 1.5 times. Although both chemical signal and structure signal shows similar effects on activating paracrine effects between HDFs and HUVECs, the paracrine effects stimulated by combined biomaterial signals are significantly higher than that of the simple addition of the single signal stimulation, suggesting a synergetic effect of the combined signals on the expression of downstream angiogenic molecules in co-HUVEC. To confirm the angiogenic effect of the combined biomaterial signals, cells co-cultured on different substrates with different media for 3 days were stained with vWF antibody (Fig. 1d). The results demonstrated that structural signals of electrospun scaffolds consisting of random or aligned nanofibers significantly enhanced the tube formation of HUVECs in co-cultures as compared to non-structure control, and the tubes formed on electrospun scaffolds with aligned nanofibers were much denser and longer than those formed on electrospun scaffolds with random nanofibers. Meanwhile, as for the effects of chemical signals, medium containing 1/128BG also enhanced the tube formation of HUVECs in co-cultures as compared to control medium. Furthermore, the combined structural signals of aligned nanofibers and chemical signals of 1/128BG revealed a further stimulatory effect on tube formation of HUVECs in co-cultures as compared to the single stimulation such as structural or chemical signal. 3.3. Effects of combined biomaterial signals on matrix synthesis in HDF-HUVEC co-culture Extracellular matrix synthesis is one of the key steps in the wound healing process. To test the effects of combined biomaterial signals on co-cultured HDFs in the co-culture system, the expression of collagen I, elastin and fibronectin in HDF-HUVEC cocultures under different conditions were measured. When cells were cultured with different structural signals but same chemical signals, as compared to control group, electrospun scaffolds showed stimulatory effects on expression of collagen, elastin and fibronectin in co-HDF (Fig. 2a–c). In addition, the electrospun scaffolds with aligned nanofibers showed higher stimulatory effects than the electrospun scaffolds with random nanofibers, as the former upregulated expression of the three genes in co-HDF for about 2 times while the latter upregulated those gene expressions for only about 1.5 times. Meanwhile, when cells were cultured with same structural signals, the expression of collagen I, elastin and fibronectin in co-HDF cultured with 1/128BG medium were about 1.5 times higher than those in co-HDF cultured with medium without BG ionic products. It is interesting to see that the combination of aligned nanofibers and 1/128BG enhanced the synthesis of

Please cite this article in press as: Y. Xu et al., Combined chemical and structural signals of biomaterials synergistically activate cell-cell communications for improving tissue regeneration, Acta Biomater. (2017), http://dx.doi.org/10.1016/j.actbio.2017.03.056

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Fig. 1. Combined biomaterial signals promote vascular growth factor secretion in HDF-HUVEC co-cultures. (a–c) Gene expression of VEGF, KDR and eNOS from monocultured cells and separated co-HUVEC and co-HDF cultured under different conditions. ⁄represents p < 0.05, and ⁄⁄ represents p < 0.01 when the data were compared with cells cultured on coverslips (control structure) in control medium, n = 3. D represents p < 0.05, and DD represents p < 0.01 when the data were compared with aligned structure in control medium, n = 3.% represents p < 0.05, and %% represents p < 0.01 when the data were compared with aligned structure in BG containing medium, n = 3. (d) vWF staining images of HDF-HUVEC co-cultured on electrospun scaffolds with different structures in control medium and BG extracts diluted at 1/128 for 3 days. Scale bar = 200 lm.

extracellular matrix protein synthesis in co-HDF for about 10 times as compared to the control group (Fig. 2a–c), which is much higher than the single stimulatory effect of pure 1/128BG or pure electrospun aligned nanofibers or the simple addition of both effects, indicating the synergetic effects of the chemical signal of BG ionic products and structural signal of electrospun nanofibers on matrix synthesis in HDF-HUVEC co-cultures. The effects of biomaterial signals on gene and protein expression of collagen I, fibronectin and elastin were similar (Fig. 2d–f).

3.4. Effects of combined biomaterial signals on cell-cell communications in HDF-HUVEC co-culture In order to confirm the effects of combined chemical and structural signals of biomaterials on cell-cell interactions, we further evaluated the cell-cell communication by analyzing the expression

of the gap junction protein of Cx43 in HDF-HUVEC co-cultures. Considering the observed paracrine effects between HUVECs and HDFs in the co-culture system where the co-cultured HDFs mainly expressed VEGF while the co-cultured HUVECs mainly expressed VEGF receptor 2 KDR and eNOS, we assumed that the communication between HDFs and HUVECs in the co-culture system might contribute to the synergetic stimulatory effects of combined signals. Fig. 3 shows the expression of gap junctional molecule Cx43 in HDF-HUVEC co-cultures. When the cells were cultured on different substrates with same culture medium, as compared to the control group, electrospun scaffolds with aligned nanofibers significantly upregulated Cx43 gene expression for about 2.5 times in co-HDF and 3 times in co-HUVEC while electrospun scaffolds with random nanofibers only increased the expression of Cx43 for about 2 times in co-HDF and about 2.5 times in co-HUVEC, indicating that structural signals significantly activated cell-cell com-

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Fig. 2. Combined biomaterial signals promote matrix synthesis in HDF-HUVEC co-cultures. (a–c) Gene expression of collagen I, elastin and fibronectin from mono-cultured cells and separated co-HUVEC and co-HDF cultured under different conditions, respectively. ⁄ represents p < 0.05, and ⁄⁄ represents p < 0.01 when the data were compared with cells cultured on coverslips in control medium, n = 3. D represents p < 0.05, and DD represents p < 0.01 when the data were compared with aligned structure in control medium, n = 3.% represents p < 0.05, and%% represents p < 0.01 when the data were compared with aligned structure in BG containing medium, n = 3. (d–f) Immunofluorescence staining images of collagen I, elastin and fibronectin in HDF-HUVEC co-cultured under different conditions. Scale bar = 100 lm.

munication by stimulating Cx43 expression. However, when cells were cultured on same substrates, 1/128BG medium only increased the expression of Cx43 in co-HUVEC for about 1.2–1.5 times as compared to 0BG, but did not significantly increased the expression of Cx43 in co-HDF. This result indicates that the structural signals of aligned nanofibers played a more important role in stimulating cell-cell communications than chemical signals of BG ionic products. More interesting to see is that, although the increase of Cx43 expression in the co-cultured cells stimulated by combined signals (about 2.5–3.5 times) is much higher than that in the co-cultured cells simulated only by 1/128BG (about 1.2 times) or aligned nanofibers (about 2–2.5 times) alone, the two stimulatory signals show only simple superposition effects on cell-cell communications through gap junctional molecule Cx43 and no synergetic effects between the two biomaterial signals were observed. The effect of biomaterial signals on gene and protein expression of Cx43 was similar. 3.5. Effects of combined biomaterial signals on morphology of endothelial cells and fibroblasts To understand why the structural signals of aligned nanofibers show higher stimulatory effects on Cx43 expression in HDF-HUVEC co-culture, we observed the morphology and distribution of

HUVECs and HDFs cultured under different conditions. The results revealed that HDFs or HUVECs cultured on electrospun scaffolds with aligned structure elongated along the direction of nanofibers, while those cells cultured on electrospun scaffolds with random structure showed random arrangement (Fig. 4a–b). However, when the cells were cultured on same substrates with different culture media (0BG or 1/128BG), there is no obvious difference in distribution or arrangement of HUVECs or HDFs, indicating that chemical signals of biomaterials have less effects on the morphology and alignment of cells than the structural signals. 3.6. Skin tissue engineering constructs for treatment of wound healing 3.6.1. Wound closure To confirm the in vivo function of the new skin tissue engineering constructs, the constructs of HDFs and HUVCEs co-culture/ electrospun scaffolds activated by aligned nanofibers structure and bioactive ions of BG extracts diluted at 1/128 ratio were applied in a chronic wound healing model. Macroscopic analysis and closure measurements showed that the new co-culture construct activated by aligned electrospun fibrous structure and bioactive BG ions significantly enhanced wound closure as compared to other groups, while the co-culture constructs activated only by aligned electrospun fibrous structure, bioactive BG ions or without

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Fig. 3. Combined biomaterial signals improve gap junctional communications in HDF-HUVEC co-cultures. (a) Gene expression of Cx43 from mono-cultured cells and separated co-HUVEC and co-HDF cultured under different conditions. ⁄ represents p < 0.05 when the data were compared with cells cultured on same substrate in control medium, n = 3. D represents p < 0.05, and DD represents p < 0.01 when the data were compared with aligned structure in control medium, n = 3.% represents p < 0.05, and%% represents p < 0.01 when the data were compared with aligned structure in BG containing medium, n = 3. (b) Immunofluorescence staining of Cx43 in HDF-HUVEC cocultured under different conditions. Scale bar = 100 lm.

Fig. 4. Combined biomaterial signals affect morphology of endothelial cells and fibroblasts. (a) F-actin staining images of HUVECs cultured on electrospun scaffolds with different structures in control medium and BG containing medium at dilution of 1/128 for 3 days. Scale bar = 100 lm. (b) F-actin staining images of HDFs cultured on electrospun scaffolds with different structures in control medium and BG containing medium at dilution of 1/128 for 3 days. Scale bar = 100 lm.

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any stimulation also showed certain degree of enhancement as compared with the positive and negative controls (Fig. 5a–b). The wound closure percentage for the electrospun 1/128BG coculture construct group was 52.15% ± 3.63 at day 7 and 98.17% ± 1.79 at day 14 after surgery, which were significantly higher than those in other groups (Fig. 5b). Wound healing was further analyzed by HE staining and the results are shown in Fig. 5c. It can be seen that the co-culture construct activated by aligned electrospun fibrous structure and bioactive BG ions performed the most improved enhancement of wound healing among all groups, as it showed the thickest eschar at day 7 and thickest newly formed neoepidermis at day 14, while the coculture construct activated by single aligned electrospun fibrous structure or bioactive BG ions also promoted the formation of new neoepidermis to some degree as compared to positive and negative control groups.

3.6.2. Neovascularization and collagen I deposition in wound healing Blood vessel formation and extracellular matrix deposition are critical steps for chronic wound healing. In this study, new blood vessels in wound site treated with different conditions were observed at day 14 by CD31 immunostaining, and the deposition of collagen I were investigated by immunohistochemistry staining at day 7.

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As shown in Fig. 6a and b, the co-culture constructs activated by aligned electrospun fibrous structure and bioactive BG ions significantly stimulated neovascularization when compared with other groups, as it showed the highest blood vessel density (34 ± 3.6 per observation field) among all groups. Meanwhile, the coculture constructs activated by single aligned electrospun fibrous structure or bioactive BG ions also revealed stimulatory effects on neovascularization. In addition, the extracellular matrix deposition, such as collagen I, was also significantly enhanced by the biomaterial activated co-culture construct, with the highest activation of the combination of aligned electrospun fibrous structure and bioactive BG ions (Fig. 6c) as compared with the controls.

4. Discussion For a long time, the tissue engineering strategy has been involving only one type of cells, and biomaterials have been only used as scaffolds to physically support cell growth. However, single cell type is far from the real environment of the tissue regeneration process, and biomaterial characteristics, such as material chemistry and structure, with which cells and tissues will be in contact in the microenvironment during tissue regeneration, may also have biological activity to actively affect cell behaviors and tissue regeneration processes. Recently, co-culture systems of different

Fig. 5. Combined biomaterial signals promote wound closure and neoepidermis formation in wound healing. (a) Wound healing condition at day 0, 2, 7, and 14 after surgery. (b) Wound closure percentage in all groups, n = 3. ⁄ represents p < 0.05, and ⁄⁄ represents p < 0.01 as compared the data with electrospun 1/128BG co-culture construct. (c) HE staining of sections from tissue samples of mouse excision wounds after the treatment with different conditions for 2, 7, and 14 days. ES = eschar, NE = neoepidermis, GT = granulation tissue, ND = neodermis. Scale bar = 200 lm.

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Fig. 6. Combined biomaterial signals promote neovascularization and deposition of collagen I in wound healing. (a) New blood vessels immunohistochemistry stained with CD31 (arrow heads) in tissue samples after mouse excision wounds treated with different conditions for 14 days. Scale bar = 200 lm. (b) Statistical analysis of vessel density at 14 days after surgery. The vessel density is the number of vessels in a field in the upright microscope with a magnification of 200. ⁄ represents p < 0.05, and ⁄⁄ represents p < 0.01 as compared the data with electrospun 1/128 co-culture constructs. n = 3. (c) Immunohistochemistry staining of collagen I in wound tissues at day 7 after surgery. Scale bar = 200 lm.

cell types have been applied in order to closely mimic the in vivo cellular microenvironment of tissue regeneration [38–41]. Meanwhile, more and more studies revealed that biomaterials may stimulate the behavior of specific types of cells related to tissue regeneration by their bioactive components or special structures [42–44]. Our recent studies have demonstrated that bioactive ions released from biomaterials can even stimulate cell-cell interactions between different types of cells, which more significantly enhanced vascularization than one single type of cells or cocultures without any biomaterial stimulation [18,19]. Therefore, in order to better mimic the tissue regeneration process in which biomaterials are involved, both co-culture of different types of cells and combined biomaterial signals should be considered in tissue engineering. We propose that optimal design of bioactive materials with controllable bioactive chemical and structural signals is critical, and elucidating the mechanism of combined biomaterial signals on co-cultured cells will lead to an improved tissue engineering strategy, which can much more closely mimic the cellular environment in tissue regeneration process. To prove our concept, in the present study, we for the first time demonstrated that the novel construct of co-cultured fibroblasts and endothelia cells on biomaterial scaffolds activated by unique aligned nanofiber structure and bioactive ions significantly enhanced chronic wound healing as compared to non-activated co-cultured cells. Obviously, considering the multiple cellular environment and interaction of biomaterials and cells during tissue regeneration, the biomaterial activated co-culture skin tissue engineering con-

struct designed in this study can much better mimic the in vivo complex microenvironment than conventional skin tissue engineering constructs. However, the combined effect of chemical and structural signals on cells has not been well explored so far. The fundamental question first to answer is what kind of biomaterial signal stimulates cell behaviors and cell-cell interactions. Specifically, whether the combination of chemical and structural biomaterial signals has synergistic effect on tissue regeneration related cell behaviors and cell-cell interactions? Our results first confirmed that both single chemical and structural signals of biomaterials can stimulate cell-cell interactions, and both nanofibrous structure of electrospun scaffolds and bioactive ions released from BG stimulated interactions between HDFs and HUVECs by activating paracrine effects and gap junctional communication, and subsequently upregulated vascular growth factor expression and extracellular matrix protein deposition. More interestingly, our results further demonstrated that the chemical signals of BG ions and structural signals of aligned nanofibers showed rather synergistic than superposition effects on stimulating the expression of vascular growth factors, vascularization and protein synthesis in HDF-HUVEC co-cultures. Si ions contribute significantly to the chemical signals of BG. The most effective Si ion concentrations (0.88 mg/ml in ECM with 1/128BG and 1.14 mg/ml in DMEM with 1/128BG) are consistent with the concentrations obtained in our previous studies where the most effective concentrations of Si ion for stimulating wound healing were in the range of 0.6– 1.7 mg/ml [18,19].

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The second fundamental question is how the biomaterial signals influence cell behaviors and cell-cell interactions. It is interesting that, although the two stimulatory signals of biomaterials show synergistic stimulatory effects on the cell-cell communications in co-culture cell system, each signal has its specific role. For stimulating cell-cell interactions through gap junction, the structural signal of aligned nanofibers plays more important role than the chemical signal of BG ionic products. Researchers have found that topography of electrospun nanofibers can change the cell cytoskeleton with the observations of actin microfilament alignment [45–47] and subsequent cell differentiation as the formation of adhesion plaques and the development of cell cytoskeleton are of great importance to cell differentiation [48,49]. In the current study, we found that the structural signals of nanofibers in electrospun scaffolds significantly affected the morphology of HDFs and HUVECs while BG ionic products did not. Characterization of electrospun scaffolds shows that nanofibers in electrospun scaffolds with aligned structure oriented in one direction while those in electrospun scaffolds with random structure distributed randomly when there is no difference in the wettability of the two electrospun scaffolds. Therefore, the electrospun scaffolds with aligned nanofibers may remodel the skeletons and nuclei of cells and guide cells along the direction of nanofibers, which results in a much closer cell-cell contact compared to the cells distributing randomly, and finally leads to promoted communications between cells through gap junction and cell differentiation [50,51]. However, for stimulating cell-cell communication through paracrine effects, both the chemical signals of BG ionic products and structural signals of aligned nanofibers showed similar stimulatory degree on the expression of vascular growth factors as well as vascularization. It seems that the spatial distribution and shape of cells are not critical for the paracrine effect as it performs through soluble growth factors released by the cells. It has been reported that BG ionic products can directly influence signal transduction by modulating chemical signals in processes of cell metabolism, leading to long-term effects on cell differentiation [52]. Taken together, the mechanisms through which chemical signals of bioactive ions and structural signals of electrospun nanofibers affecting cell behaviors and cell-cell interactions are different. The ions released from BG may participate directly in cell metabolism to modulate the activities of cells while electrospun nanofibers with different structures affect cell behaviors by remodeling cytoskeleton and nuclei of cells. In addition, the chemical signals of BG ionic products mainly stimulate the cell-cell communications through enhancing paracrine effects while the structural signals of aligned nanofibers stimulate the cell-cell communications through both paracrine effects and gap junction activation. The different activation mechanisms might be one of the reasons for the synergistic effects of two different biomaterial signals. To further confirm the novel tissue engineering approach utilizing co-cultured cell system and combined bioactive signals of biomaterials, we chose wound healing as a model and applied electrospun scaffolds with specific structure and bioactive ions of BG, which have been used as wound dressing [53–55], to evaluate the effectiveness of the novel constructs. Fibroblast-endothelial cell co-culture systems have been demonstrated to have better wound healing ability than fibroblasts or endothelial cells alone because the communications between the two types of cells in the co-culture system can enhance blood vessel formation in wound area [56,57]. Our results demonstrated that the HDFHUVEC co-cultured on aligned nanofibers in bioactive BG ion environment as the novel skin tissue engineering construct significantly accelerated wound healing by stimulating blood vessel formation and ECM deposition in the wound site, as compared to the wound treated by co-cultured cells without biomaterial stimulation or only stimulated with single type of biomaterial signal. As

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the BG ionic products and aligned nanofibers can synergistically upregulated the expression of angiogenic growth factors and extracellular matrix proteins in co-cultured HDFs to a greatest extent, the secreted angiogenic growth factors could further induce endothelial cells to migrate into wound site [58]. Then, angiogenesis in wound site was enhanced and wound healing process was accelerated [59]. Meanwhile, it has been reported that, in the early phase of wound healing, fibroblasts activated by BG could migrate into wound site to synthesize an extracellular matrix as the bed for newly formed blood vessels, which extremely advances the wound healing process [60,61]. In previous studies, various tissue-engineered skin substitutes have been designed. In general, they can be divided into three categories. The first category is non-cellular polymer scaffolds. In this type of tissue-engineered skin substitutes, natural, synthetic and composite polymers have been fabricated into scaffolds with different structures for skin tissue engineering [62], such as poly(lactic acid)-collagen and poly(lactic acid)-gelatin hybrid scaffolds with funnel-like porous structure [63]. Basically, the non-cellular skin substitutes just play the role of wound dressing to create a humid and sterilized healing environment, but do not have ability to promote wound healing initiatively. As a result, these substitutes can just temporarily replace the skin epidermis [21,64]. The second category is tissue-engineered skin substitutes only containing cells, mainly cell sheets without scaffolds [29], in which the migrated cells and the growth factors released from cell sheets can stimulate wound healing spontaneously [65,66]. However, the effects of biomaterials are totally ignored in this kind of substitute. Finally, the third category is tissue-engineered skin substitutes containing biomaterial scaffolds and cells. In these skin substitutes, the stimulatory effects of biomaterial scaffolds and bioactivity of cells can persistently affect wound tissue for a period of time and stimulate wound healing [58,67,68]. However, most of this kind of skin substitutes only focus on the effects of single type of biomaterial signals on single type of cells, but ignored the interactions between different types of biomaterial signals and interactions between different types of cells. As compared to these tissueengineered skin substitutes, in this study, we fabricated an improved skin tissue engineering construct, in which more than one type of bioactive biomaterial signals were combined to regulate the interactions between critical cells involved in wound healing. The elaborately designed skin tissue engineering construct took advantages of the different biomaterial stimulatory effects on cell-cell interactions and showed higher efficacy in stimulating wound healing as compared to other tissue engineering constructs. In summary, the underline mechanism for enhancement of chronic wound healing by the novel skin constructs is that the structural signal of electrospun aligned nanofibers and the chemical signal of bioactive ions from BG synergistically stimulated the cell-cell interactions in the HDF-HUVEC co-cultures via activating paracrine effects and gap junctional communication between two cells, which results in upregulated vascular growth factor expression, vascularization and matrix protein synthesis (Fig. 7). Our new concept of the co-culture tissue engineering construct activated by multiple bioactive signals of biomaterials showed great potential not only for wound healing applications but also for tissue engineering of other type of tissues. In this study, to prove our concepts, HDFs and HUVECs were co-cultured on electrospun nanofibers with cell culture media containing BG ionic products. However, immune deficient animal model was used for the in vivo study, which is different to the clinical situations. Therefore, the clinical application potential of the approach needs to be further confirmed in animal models with normal immune system. In addition, for practical applications, a biomaterial system containing both of bioactive structural and chemical signals, which

Please cite this article in press as: Y. Xu et al., Combined chemical and structural signals of biomaterials synergistically activate cell-cell communications for improving tissue regeneration, Acta Biomater. (2017), http://dx.doi.org/10.1016/j.actbio.2017.03.056

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References

Fig. 7. The underline mechanism for enhancement of chronic wound healing by the novel skin constructs.

is suitable for co-culturing two or more types of cells, needs to be designed.

5. Conclusion In this study, we demonstrated that, as compared to single biomaterial signals such as structural signals of electrospun aligned nanofibers or chemical signals of bioactive ions in BG extracts, combination of these two type of signals showed synergistic stimulatory effects on differentiation of endothelial cells or fibroblasts by through stimulating paracrine effects and junctional communications between HDFs and HUVECs, resulting upregulated expression of angiogenic growth factors in endothelial cells and matrix proteins in fibroblasts. Paracrine effects were stimulated by both the chemical signals of BG ionic products and the structural signals of aligned nanofibers while gap junctional communication was mainly activated by the structural signals, which may contribute to the synergistic stimulatory effects of the combined signals. Skin tissue constructs containing HDF-HUVEC co-cultures activated by the combined signals could better enhance wound healing through stimulating angiogenesis and collagen I deposition in wound site. These results suggest that the combination of different stimulatory signals and co-cultures of critical cells may be a promising and effective approach for tissue engineering.

Ethical standards The authors declare that all of the experiments comply with the current laws of China.

Acknowledgements This work was supported by Natural Science Foundation of China (Grant No.: 31470918), the National Key Research and Development Program of China (Grant No. 2016YFC1100201), Medicine-Engineering Cross-Research Foundation of Shanghai Jiao Tong University (YG2015ZD06), and the 2016 ‘‘SMC-Chenxing” Talent Program of Shanghai Jiao Tong University.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2017.03. 056.

[1] R. Langer, Tissue engineering, Science 260 (1993) 920–926. [2] R. Langer, D.A. Tirrell, Designing materials for biology and medicine, Nature 428 (2010) 487–492. [3] J.M. Sorrell, M.A. Baber, A.I. Caplan, A self-assembled fibroblast-endothelial cell co-culture system that supports in vitro vasculogenesis by both human umbilical vein endothelial cells and human dermal microvascular endothelial cells, Cells Tissues Organs 186 (2007) 157–168, http://dx.doi.org/10.1159/ 000106670. [4] M. Grellier, L. Bordenave, J. Amedee, Cell-to-cell communication between osteogenic and endothelial lineages: implications for tissue engineering, Trends Biotechnol. 27 (2009) 562–571. 10.1016/j.tibtech.2009.07.001. [5] M.N. Rahaman, D.E. Day, B.S. Bal, Q. Fu, S.B. Jung, L.F. Bonewald, A.P. Tomsia, Bioactive glass in tissue engineering, Acta Biomater. 7 (2011) 2355–2373. 10.1016/j.actbio.2011.03.016. [6] A. Hoppe, N.S. Güldal, A.R. Boccaccini, A review of the biological response to ionic dissolution products from bioactive glasses and glass-ceramics, Biomaterials 32 (2011) 2757–2774. [7] G. Jell, M.M. Stevens, Gene activation by bioactive glasses, J. Mater. Sci. – Mater. Med. 17 (2006) 997–1002. [8] S. Ni, J. Chang, L. Chou, A novel bioactive porous CaSiO3 scaffold for bone tissue engineering, J. Biomed. Mater. Res. A 76 (2006) 196–205, http://dx.doi.org/ 10.1002/jbm.a.30525. [9] C. Wang, K. Lin, J. Chang, J. Sun, Osteogenesis and angiogenesis induced by porous beta-CaSiO(3)/PDLGA composite scaffold via activation of AMPK/ERK1/ 2 and PI3K/Akt pathways, Biomaterials 34 (2013) 64–77, http://dx.doi.org/ 10.1016/j.biomaterials.2012.09.021. [10] W.J. Li, R. Tuli, X. Huang, P. Laquerriere, R.S. Tuan, Multilineage differentiation of human mesenchymal stem cells in a three-dimensional nanofibrous scaffold, Biomaterials 26 (2005) 5158–5166. [11] M.J. Dalby, N. Gadegaard, R. Tare, A. Andar, M.O. Riehle, P. Herzyk, C.D.W. Wilkinson, R.O.C. Oreffo, The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder, Nat. Mater. 6 (2007) 997–1003. [12] A.A. Gorustovich, J.A. Roether, A.R. Boccaccini, Effect of bioactive glasses on angiogenesis: a review of in vitro and in vivo evidences, Tissue Eng. Part B Rev. 16 (2010) 199–207. [13] C.J. Shih, P.S. Lu, C.H. Hsieh, W.C. Chen, J.C. Chen, Effects of bioglass powders with and without mesoporous structures on fibroblast and osteoblast responses, Appl. Surf. Sci. 314 (2014) 967–972. [14] G. Liao, L. Chen, X. Zeng, X. Zhou, X. Xie, E. Peng, Z. Ye, Y. Mai, Electrospun poly (L-lactide)/poly(e-caprolactone) blend fibers and their cellular response to adipose-derived stem cells, J. Appl. Polym. Sci. 120 (2010) 2154–2165. [15] H. Xu, W. Cui, J. Chang, Fabrication of patterned PDLLA/PCL composite scaffold by electrospinning, J. Appl. Polym. Sci. 127 (2013) 1550–1554. [16] P. Uttayarat, A. Perets, M. Li, P. Pimton, S.J. Stachelek, I. Alferiev, R.J. Composto, R.J. Levy, P.I. Lelkes, Micropatterning of three-dimensional electrospun polyurethane vascular grafts, Acta Biomater. 6 (2010) 4229–4237. [17] R.B. Montero, X. Vial, D.T. Nguyen, S. Farhand, M. Reardon, M.P. Si, G. Tsechpenakis, F.M. Andreopoulos, BFGF-containing electrospun gelatin scaffolds with controlled nano-architectural features for directed angiogenesis, Acta Biomater. 8 (2012) 1778–1791. [18] H. Li, J. Chang, Bioactive silicate materials stimulate angiogenesis in fibroblast and endothelial cell co-culture system through paracrine effect, Acta Biomater. 9 (2013) 6981–6991, http://dx.doi.org/10.1016/j.actbio.2013.02.014. [19] H. Li, K. Xue, N. Kong, K. Liu, J. Chang, Silicate bioceramics enhanced vascularization and osteogenesis through stimulating interactions between endothelia cells and bone marrow stromal cells, Biomaterials 35 (2014) 3803– 3818, http://dx.doi.org/10.1016/j.biomaterials.2014.01.039. [20] L. Ma, C. Gao, Z. Mao, J. Zhou, J. Shen, X. Hu, C. Han, Collagen/chitosan porous scaffolds with improved biostability for skin tissue engineering, Biomaterials 24 (2003) 4833–4841, http://dx.doi.org/10.1016/S0142-9612(03)00374-0. [21] S.G. Kumbar, S.P. Nukavarapu, R. James, L.S. Nair, C.T. Laurencin, Electrospun poly(lactic acid-co-glycolic acid) scaffolds for skin tissue engineering, Biomaterials 29 (2008) 4100–4107. [22] K. Lindberg, S.F. Badylak, Porcine small intestinal submucosa (SIS): a bioscaffold supporting in vitro primary human epidermal cell differentiation and synthesis of basement membrane proteins, Burns 27 (2001) 254–266. [23] R.H. Demling, L. Desanti, Management of partial thickness facial burns (comparison of topical antibiotics and bio-engineered skin substitutes), Burns 25 (1999) 256–261. [24] F. Groeber, M. Holeiter, M. Hampel, S. Hinderer, K. Schenke-Layland, Skin tissue engineering–in vivo and in vitro applications, Adv. Drug Deliv. Rev. 63 (2011) 352. [25] S. Intaraprasit, A. Faikrua, A. Sittichokechaiwut, J. Viyoch, Efficacy evaluation of the fibroblast-seeded collagen/chitosan scaffold on application in skin tissue engineering, Science Asia 38 (2012) 268. [26] A. Lorenti, Wound healing: from epidermis culture to tissue engineering, CellBio 1 (2012) 17–29. [27] E. Petersen, Realization feature of mesenchymal dermal cells tissue engineering construction response in granulating wound transplantation in relation with time-frame, J. Cosmet. Dermatol. Sci. Appl. 2 (2012) 126–129. [28] M.E. Balañá, H.E. Charreau, G.J. Leirós, Epidermal stem cells and skin tissue engineering in hair follicle regeneration, World J. Stem Cells 7 (2015) 711.

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Y. Xu et al. / Acta Biomaterialia xxx (2017) xxx–xxx [29] M.T. Cerqueira, R.P. Pirraco, A.R. Martins, T.C. Santos, R.L. Reis, A.P. Marques, Cell sheet technology-driven re-epithelialization and neovascularization of skin wounds, Acta Biomater. 10 (2014) 3145. [30] C.J. Angammana, S.H. Jayaram, The effects of electric field on the multijet electrospinning process and fiber morphology, IEEE Trans. Ind. Appl. 47 (2011) 1028–1035. [31] H. Li, J. Chang, Fabrication and characterization of bioactive wollastonite/PHBV composite scaffolds, Biomaterials 25 (2004) 5473–5480, http://dx.doi.org/ 10.1016/j.biomaterials.2003.12.052. [32] C. Wu, C. Jiang, W. Zhai, S. Ni, J. Wang, Porous akermanite scaffolds for bone tissue engineering: preparation, characterization, and in vitro studies, J. Biomed. Mater. Res. Part B Appl. Biomater. 78 (2006) 47–55. [33] L. Bordenave, C. Baquey, R. Bareille, F. Lefebvre, C. Lauroua, V. Guerin, F. Rouais, N. More, C. Vergnes, J.M. Anderson, Endothelial cell compatibility testing of three different Pellethanes, J. Biomed. Mater. Res. 27 (1993) 1367–1381. [34] J.M. Sorrell, M.A. Baber, L. Brinon, D.A. Carrino, M. Seavolt, D. Asselineau, A.I. Caplan, Production of a monoclonal antibody, DF-5, that identifies cells at the epithelial-mesenchymal interface in normal human skin. APN/CD13 is an epithelial-mesenchymal marker in skin, Exp. Dermatol. 12 (2003) 315–323. [35] T. Okano, N. Yamada, M. Okuhara, H. Sakai, Y. Sakurai, Mechanism of cell detachment from temperature-modulated, hydrophilic-hydrophobic polymer surfaces, Biomaterials 16 (1995) 297–303. [36] R.K. Brazzell, M.E. Stern, J.V. Aquavella, R.W. Beuerman, L. Baird, Human recombinant epidermal growth factor in experimental corneal wound healing, Invest. Ophthalmol. Vis. Sci. 32 (1991) 336–340. [37] K. Young-Bae, K. Hyun-Woo, R. Dae-Hyun, Y. Seo-Yeon, B. Rong-Min, K. JeumYong, K. Haeyong, L. Kwang-Gill, P. Young-Hwan, L. Jang-Hern, Topical application of epidermal growth factor accelerates wound healing by myofibroblast proliferation and collagen synthesis in rat, J. Vet. Sci. 7 (2006) 105–109. [38] H. Yu, P.J. Vandevord, L. Mao, H.W. Matthew, P.H. Wooley, S.Y. Yang, Improved tissue-engineered bone regeneration by endothelial cell mediated vascularization, Biomaterials 30 (2008) 508–517. [39] S. Fuchs, A. Hofmann, C. Kirkpatrick, Microvessel-like structures from outgrowth endothelial cells from human peripheral blood in 2-dimensional and 3-dimensional co-cultures with osteoblastic lineage cells, Tissue Eng. 13 (2007) 2577–2588. [40] C.J. Kirkpatrick, S. Fuchs, R.E. Unger, Co-culture systems for vascularization — learning from nature, Adv. Drug Deliv. Rev. 63 (2011) 291–299, http://dx.doi. org/10.1016/j.addr.2011.01.009. [41] D. Kaigler, P.H. Krebsbach, E.R. West, K. Horger, Y.C. Huang, D.J. Mooney, Endothelial cell modulation of bone marrow stromal cell osteogenic potential, Faseb J. Off. Publ. Fed. Am. Soc. Exp. Biol. 19 (2005) 665–667. [42] L.-C. Gerhardt, A.R. Boccaccini, Bioactive glass and glass-ceramic scaffolds for bone tissue engineering, Materials (Basel) 3 (2010) 3867–3910, http://dx.doi. org/10.3390/ma3073867. [43] S. Patel, K. Kurpinski, R. Quigley, H. Gao, B.S. Hsiao, M.-M. Poo, S. Li, Bioactive nanofibers: synergistic effects of nanotopography and chemical signaling on cell guidance, Nano Lett. 7 (2007) 2122–2128. [44] H. Li, Y. Xu, H. Xu, J. Chang, Electrospun membranes: control of the structure and structure related applications in tissue regeneration and drug delivery, J. Mater. Chem. B 2 (2014) 5492–5510. [45] M.J. Dalby, M.O. Riehle, S.J. Yarwood, C.D.W. Wilkinson, A.S.G. Curtis, Nucleus alignment and cell signaling in fibroblasts: response to a micro-grooved topography, Exp. Cell Res. 284 (2003) 272–280. [46] Y. Lei, O.F. Zouani, M. Remy, C. Ayela, M.C. Durrieu, Geometrical microfeature cues for directing tubulogenesis of endothelial cells, PLoS One 7 (2012) e41163. [47] H. Xu, H. Li, Q. Ke, J. Chang, An anisotropically and heterogeneously aligned patterned electrospun scaffold with tailored mechanical property and

[48] [49]

[50] [51] [52]

[53]

[54]

[55]

[56] [57]

[58]

[59] [60] [61]

[62] [63]

[64]

[65] [66]

[67]

[68]

13

improved bioactivity for vascular tissue engineering, ACS Appl. Mater. Interfaces 7 (2015) 8706–8718, http://dx.doi.org/10.1021/acsami.5b00996. R.L. Juliano, S. Haskill, Signal transduction from the extracellular matrix, J. Cell Biol. 120 (1993) 577. A.E. Aplin, A. Howe, S.K. Alahari, R.L. Juliano, Signal transduction and signal modulation by cell adhesion receptors: the role of integrins, cadherins, immunoglobulin-cell adhesion molecules, and selectins, Pharmacol Rev. 50 (1998) 197–263. J.P. Stains, R. Civitelli, Gap junctions in skeletal development and function, Biochim. Biophys. Acta 1719 (2006) 69–81. H.A. Dbouk, Connexins: a myriad of functions extending beyond assembly of gap junction channels, Cell Commun. Signal 7 (2009) 1–17. I.D. Xynos, A.J. Edgar, L.D.K. Buttery, L.L. Hench, J.M. Polak, Ionic products of bioactive glass dissolution increase proliferation of human osteoblasts and induce insulin-like growth factor II mRNA expression and protein synthesis, Biochem. Biophys. Res. Commun. 276 (2000) 461–465. S. Jebahi, H. Oudadesse, N. Jardak, I. Khayat, H. Keskes, A. Khabir, T. Rebai, F.H. El, F.A. El, Biological therapy of strontium-substituted bioglass for soft tissue wound-healing: responses to oxidative stress in ovariectomised rats, Ann. Pharm. Françaises 71 (2013) 234. H. Xu, F. Lv, Y. Zhang, Z. Yi, Q. Ke, C. Wu, M. Liu, J. Chang, Hierarchically micropatterned nanofibrous scaffolds with a nanosized bio-glass surface for accelerating wound healing, Nanoscale 7 (2015) 18446–18452. E.J. Chong, T.T. Phan, I.J. Lim, Y.Z. Zhang, B.H. Bay, S. Ramakrishna, C.T. Lim, Evaluation of electrospun PCL/gelatin nanofibrous scaffold for wound healing and layered dermal reconstitution, Acta Biomater. 3 (2007) 321–330. J. Li, J. Chen, R. Kirsner, Pathophysiology of acute wound healing, Clin. Dermatol. 25 (2007) 9–18. Y.K. Jeon, Y.H. Jang, D.R. Yoo, S.N. Kim, S.K. Lee, M.J. Nam, Mesenchymal stem cells’ interaction with skin: wound-healing effect on fibroblast cells and skin tissue, Wound Repair Regen. 18 (2010) 655–661, http://dx.doi.org/10.1111/ j.1524-475X.2010.00636.x. H. Yu, J. Peng, Y. Xu, J. Chang, H. Li, Bioglass activated skin tissue engineering constructs for wound healing, ACS Appl. Mater. Interfaces 8 (2016) 703–715, http://dx.doi.org/10.1021/acsami.5b09853. J.P. Cooke, Nitric oxide and angiogenesis, Circulation 105 (2002) 2133–2135, http://dx.doi.org/10.1161/01.cir.0000014928.45119.73. D. Telgenhoff, B. Shroot, Cellular senescence mechanisms in chronic wound healing, Cell Death Differ. 12 (2005) 695–698. O. Stojadinovic, A. Kodra, M. Golinko, M. Tomic-Canic, H. Brem, A. Novel, Nonangiogenic, mechanism of Vegf: stimulation of keratinocyte and fibroblast migration, Wound Repair Regen. 15 (2007) A30. D. Sundaramurthi, U.M. Krishnan, S. Sethuraman, Electrospun nanofibers as scaffolds for skin tissue engineering, Polym. Rev. 54 (2014) 348–376. H. Lu, H.H. Oh, N. Kawazoe, K. Yamagishi, G. Chen, PLLA–collagen and PLLA– gelatin hybrid scaffolds with funnel-like porous structure for skin tissue engineering, Sci. Technol. Adv. Mater. 13 (2016) 64210–64218. X. Zhu, W. Cui, X. Li, Y. Jin, Electrospun fibrous mats with high porosity as potential scaffolds for skin tissue engineering, Biomacromolecules 9 (2008) 1795–1801. M.M. Mclaughlin, K.G. Marra, The use of adipose-derived stem cells as sheets for wound healing, Organogenesis 9 (2013) 79–81. Y.C. Lin, T. Grahovac, S.J. Oh, M. Ieraci, J.P. Rubin, K.G. Marra, Evaluation of a multi-layer adipose-derived stem cell sheet in a full-thickness wound healing model, Acta Biomater. 9 (2013) 5243. G. Jin, M.P. Prabhakaran, S. Ramakrishna, Stem cell differentiation to epidermal lineages on electrospun nanofibrous substrates for skin tissue engineering, Acta Biomater. 7 (2011) 3113–3122. K. Ma, S. Liao, L. He, J. Lu, S. Ramakrishna, C.K. Chan, Effects of nanofiber/stem cell composite on wound healing in acute full-thickness skin wounds, Tissue Eng. Part A 17 (2011) 1413.

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