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Bioglass activated skin tissue engineering constructs for wound healing Hongfei Yu, Jinliang Peng, Yuhong Xu, Jiang Chang, and Haiyan Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b09853 • Publication Date (Web): 18 Dec 2015 Downloaded from http://pubs.acs.org on December 27, 2015

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ACS Applied Materials & Interfaces

Bioglass activated skin tissue engineering constructs for wound healing Hongfei Yua, 1, Jinliang Penga, 2, Yuhong Xu2, Jiang Chang1, 3, Haiyan Li1* 1

Med-X Research Institute, School of Biomedical Engineering, Shanghai Jiao Tong

University, 1954 Huashan Road, Shanghai 200030, China. 2

School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road,

Shanghai 200240, China. 3

Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Dingxi Road,

Shanghai 200050, China. a

The two authors contributed to the work equally.

KEYWORDS: wound healing, bioglass, fibroblast, skin graft, angiogenesis

ABSTRACT: Wound healing is a complicated process and fibroblast is a major cell type that participates in the process. Recent studies have shown that bioglass (BG) can stimulate fibroblasts to secrete a multitude of growth factors that are critical for wound healing. Therefore, we hypothesize that BG can stimulate fibroblasts to have a higher bioactivity by secreting more bioactive growth factors and proteins as compared to untreated fibroblasts and we aim to construct a bioactive skin tissue engineering graft for wound healing by using BG activated fibroblast sheet. Thus, the effects of BG on fibroblast behaviors were studied and the bioactive skin tissue

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engineering grafts containing BG activated fibroblasts were applied to repair the full skin lesions on nude mouse. Results showed that BG stimulated fibroblasts to express some critical growth factors and important proteins including vascular endothelial growth factor, basic fibroblast growth factor, epidermal growth factor, collagen I and fibronectin. In vivo results revealed that fibroblasts in the bioactive skin tissue engineering grafts migrated into wound bed and the migration ability of fibroblasts was stimulated by BG. In addition, the bioactive BG activated fibroblast skin tissue engineering grafts could largely increase the blood vessel formation, enhance the production of collagen I, and stimulate the differentiation of fibroblasts into myofibroblasts in the wound site, which finally accelerate wound healing. This study demonstrates that the BG activated skin tissue engineering grafts contain more critical growth factors and ECM proteins that are beneficial for wound healing as compared to untreated fibroblast cell sheets.

Introduction In tissue engineering, biomaterial is one of the critical factors. It has been widely accepted that bioactive materials can affect cell behaviors during tissue engineering process

1-4

. Bioactive silicate material, such as bioglass (BG), is the first man-made

inorganic material used in bone tissue engineering because of its excellent osteostimulatory ability

5-7

. In recent years, biogalss is also used in soft tissue

engineering8. Bioglass comprises of SiO2, Na2O, CaO and P2O5 in specific proportions

9

and reactions on the surface of bioglass induce the release of soluble

ions such as Si, Ca and P. With these ionic products, BG has been reported to be able


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to strongly affect cell behaviors, in particular, stimulate osteogenic differentiation of stem cells as well as vascularization of endothelial cells (ECs), which finally stimulates osteogenesis and angiogenesis

10-11

. Recently, BG has been proved to be

able to stimulate angiogenesis of ECs and increase the secretion of angiogenic growth factors from fibroblasts, including VEGF and bFGF 10. It has been well known that these angiogenic growth factors are critically important for wound healing. Wound healing is a complicated, dynamic and interactive process, in which several kinds of cells are involved and various growth factors and proteins play important roles. According to the classic theory, wound healing can be divided into four phases: hemostasis phase, inflammatory phase, cell proliferation phase and extracellular matrix remodeling phase

12-13

. Fibroblasts and

endothelial cells proliferate and migrate in the cell proliferation phase 14. In the same stage, fibroblasts secrete angiogenic growth factors such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF)

15

. bFGF is a potent

mitogen and chemoattractant for endothelial cells and fibroblasts16 . VEGF is unique for its effects on angiogenesis, epithelialization and collagen deposition, all of which occur in the wound healing process 17. In addition to secreting of angiogenic growth factors, fibroblasts produce a new extracellular matrix (ECM) to support cell growth and form a granulation tissue bed for new capillaries and macrophages 18. Moreover, in the later phase, fibroblasts can differentiate into myofibroblasts that are responsible for wound contraction and excessive ECM deposition 19. So, fibroblasts, together with the growth factors secreted by fibroblasts, play an important role in the wound healing



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process. Since BG could stimulate fibroblasts to secret vascular growth factors, such as VEGF and bFGF, and these growth factors are important for angiogenesis

10

, we

hypothesize that BG-activated fibroblasts may be able to accelerate wound healing due to their angiogenic potential, especially for chronic wound healing. Therefore, in this study, we aim to construct a BG activated skin tissue engineering graft for wound healing by using bioglass and fibroblast as the biomaterial and cell, respectively. Through activating fibroblasts by BG to secrete critical vascular growth factors for promoting angiogenesis and to produce proteins in ECM which contribute to wound healing, the skin tissue engineering graft combining BG and fibroblasts can be used for skin regeneration, especially for chronic wound healing. Thus, in this study, we investigated the effects of BG on fibroblast behaviors with the intention to find a proper dilution ratio of BG ion products to construct a BG activated skin tissue engineering graft with fibroblasts and proper BG ionic products for enhancing wound healing. The effects of BG on fibroblast proliferation, migration and expression of growth factors and proteins were detected and optimal dilution ratio of BG ion products was selected. Then, BG activated skin tissue engineering grafts obtained through inducing fibroblast cell sheet with BG ion products with proper dilution ratio were prepared by using cell sheet technology and applied to wound healing. Fibroblast cell sheets cultured with normal cell culture medium were used as controls. The wound closure, angiogenesis, collagen deposition and myofibroblast formation in the wound site were determined.


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2. Materials and Methods 2.1 Materials BG powders used in this study were kindly provided by Shanghai Institute of Ceramics, Chinese Academy of Science. The average diameter of the BG powder particles is 20 µm (90% < 34.86 µm). BG ionic dissolution products were prepared according to the literatures

20-22

. Briefly, 1 g of BG powders were soaked in 5 ml

serum-free Dulbecco modified eagle medium (DMEM)(GIBCO) and incubated for 24 h in a humidified 37°C/5% CO2 incubator. The supernatant was collected and sterilized through a filter (Millipore, 0.22 µm) and stored at 4°C (ISO10993-1) for further use. According to our previous research findings, BG ionic dissolution products were diluted with control medium (DMEM (GIBCO) +10% fetal bovine serum (FBS) +1% penicillin–streptomycin (P/S)) at the ratios of 1/32, 1/64, 1/128, 1/256. The concentrations of various ions in terms of Ca, Si and P in these ionic dissolution products were detected by the inductively coupled plasma atomic emission spectroscopy (ICP-AES; Optima 3000DV, Perkin Elmer, USA). 2.2 Cell isolation and culture Human dermal fibroblasts (HDFs) were isolated from the superficial layer of adult human skin dermatomed at a depth of 400µm according to previous work

23

. An

Institutional Review Committee of Shanghai Jiao Tong University, School of Biomedical Engineering approved all these protocols. DMEM with 10% FBS and 1% P/S was used as HDFs’ culture medium. The culture medium was replaced every three days and HDFs at passage 3-6 were used in this study.



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2.3 Effects of BG on HDF proliferation and viability BG ionic dissolution products diluted with control medium ranging from 1/32 to 1/256 were used to culture HDFs for determining the effects of BG on HDF proliferation. The HDFs were seeded in 96-well plates at the density of 4 x 103 cells per well and were cultured in a humidified 37°C/5% CO2 incubator with control medium. After 1 day, the culture medium was replaced by the media that contained BG ionic dissolution products and cultured for 1d, 3d and 7d. The cells cultured with control medium were regarded as control. A CCK-8 assay (Cell counting kit-8, Dojindo, kumamoto, Japan) was applied to evaluate cell proliferation according the instructions. The absorbance was measured spectrophotometrically at wavelengths of 450 nm with a microplate reader (Bio-Rad Benchmark Plus), n=5. Besides, BG ionic dissolution products diluted ranging from 1/64 to 1/256 were used to detect their effects on viability of HDFs by a live-dead assay. HDFs were seeded in 24-well plates at 1 x 105 cells per well and cultured with BG containing media and control medium. After 3 days, a Live-Dead Viability Cytotoxicity kit (Invitrogen) was applied to detect cells’ viability according to the manufacturer’s instructions. 2.4 Quantitative real-time polymerase chain reaction (Q-RT-PCR) HDFs were seeded in 6-well plates at 2 x 105 cells per well and cultured in a humidified 37°C/5% CO2 incubator. BG ionic dissolution products diluted with control medium at ratios of 1/64, 1/128 and 1/256 were used to culture HDFs for 3 d and 7 d. The cells cultured with control medium were regarded as control. At the determined time point, the fibroblasts were washed twice with cold phosphate


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buffered saline (PBS) and RNA was extracted from the cells using an E.Z.N.A Total RNA kit I (OMEGA, Biotek) according to the instructions. The concentration of RNA was measured with a Nanodrop 1000 reader (Thermo Scientific) and cDNA was synthesized using a ReverTra Ace-α kit (Toyobo, Japan) according to the instructions. cDNA was diluted 1:20 with sterilized deionized water and the 4.2 µl diluted cDNA was mixed with 5.8 µl SYBR-Green and primers. The primers of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), epidermis growth factor (EGF), collagen I and fibronectin (all from Sangon Biotech (Shanghai) Co. Ltd) were used at the final concentration of 400 nM. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene. Their sequences are listed in Table1. Real-time PCR analysis was performed by using the 7900 Real-time PCR system (Applied Biosystems) in a 384-well plate. 40 cycles (95°C for 15 s, 60°C for 15 s, 72°C for 45 s) of PCR were performed after 1 min denaturation at 95°C. The data were analyzed with the SDS 2.4 software and compared by the ∆∆Ct method. Each reaction was performed in triplicate for validation. The data was then normalized to GAPDH gene expression of each condition and compared to the corresponding gene expression from control sample (cells cultured with DMEM+10% FBS+1% P/S) which were standardized to 1, n=3. 2.5 Immunofluorescence staining on fibroblasts Immunofluorescence staining of collagen I, fibronectin and α-SMA were applied to detect the production of collagen I, fibronectin, and the differentiation of fibroblasts into myofibroblasts. Briefly, HDFs were seeded on a 1-cm diameter coverslip placed



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in 24-well plates at 2 x 105 cells per well. BG ionic dissolution products diluted with control medium at the ratio 1/128 were used to culture HDFs for 3 d and the cells cultured with control medium were regarded as control. At the determined time point, the cells were washed twice with PBS and fixed with 4% (wt. /vol.) paraformaldehyde (PFA) at room temperature for 15 min. Then, the cells were permeabilized with methanol for 5 min and blocked with PBS containing 1% (wt. /vol.) bovine serum albumin (BSA) for 1 h at 37°C. After blocking, primary antibody solution containing rabbit anti-collagen I, or rabbit anti-fibronectin, or mouse anti-α-SMA (all diluted in PBS-0.5% BSA at 1/100), was added and incubated at 37°C for 12 h. To reveal collagen I, fibronectin and α-SMA, the cells were washed twice with PBS. Alexa 488 goat anti-rabbit IgG or Alexa 488 goat anti- mouse IgG secondary antibody diluted in PBS-0.5% BSA at 1/1000 was used to incubate the cells at 37°C for 1 h. Finally, the nuclei were revealed and coverslips were fixed by Prolong Gold antifade reagent with DAPI (Invitrogen) at room temperature. After being fixed, the cells were observed with a confocal microscope (Leica TCS SP5) and images were taken by a CCD camera (Leica DFC 420C). 2.6 HDF migration assay HDFs were seeded in 24-well plates at 4 x 105 cells per well and cultured in a humidified 37°C/5% CO2 incubator. After 24 h, a scratch was made with a 200 µl pipette tip at the bottom of each well followed by washing the cells with PBS for twice. Then, the culture medium was replaced by BG ionic dissolution products diluted with DMEM (without FBS) at ratio 1/128 in order to determine the effect of


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BG on HDF migration. The cells cultured with DMEM (without FBS) were regarded as control. After being cultured for 0 h and 24 h, the cells were fixed with 4% PFA for 15 min and stained with 0.05% crystal violet for 20 min at room temperature. The cells were then observed with an inverted microscope (Leica DMI 3000B, Germany) after being washed twice and images were taken with a CCD camera (Leica DFC 420C). In addition, statistical analysis of HDF migration assay was performed, we measured the original width and final width of the scratches in the two groups, the percentage of scratch shrinkage was calculated as: % of scratch shrinkage = 100×(original width - final width)/original width, n=3. 2.7 Preparation of skin grafts for wound healing In this study, cell sheet technology was used for constructing the skin grafts containing fibroblasts and BG ionic dissolution products. Cell sheet technology offers a new method in tissue regeneration without using biodegradable scaffolds and sutures

24-28

. In this technology, a thermo-responsive culture dish that can enable cell

adhesion and detachment by controllable hydrophobicity of the surface is used to obtain cell sheet skin grafts for wound healing experiment 29. At 37°C, the surface is hydrophobic but becomes hydrophilic at the temperature lower than 32°C. As a result, the cell sheet can detach from the surface and be collected

30-31

. In our study, to

prepare the bioactive skin grafts, HDFs at a density of 3 x 105 cells per dish were seeded in the commercially available Upcell 3.5 cm thermo-responsive dishes (Nunc, Thermo Scientific). BG ionic dissolution products diluted with control medium at the ratio 1/128 were used to culture HDFs. These cells were regarded as BG cell sheet.



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The cells cultured with control medium were regarded as control cell sheet. After the cells were cultured for 3 days, cell sheets were prepared according to the instructions. Briefly, the culture medium was removed and 50 µl preheated culture medium was added to prevent the cells to dry. After that, a polymembrane was placed over the cells and incubated at room temperature for 10 min for the cell sheet detachment. After being detached, the cell sheets attaching with the membrane were collected and cut into four parts for plantation. 2.8 Transplantation of the cell sheet skin grafts into full-thickness excisional wound in mouse Eighteen female nude mice (BALB/c, 6-8 weeks) were randomly divided into three groups. Three mice were used per condition and per time point (3, 7 and 14 days). Two 1 cm diameter full-thickness excisions were performed on the back of each mouse after the mouse was anaesthetized with 4% chloral hydrate. The obtained cell sheets were placed on the wound sites. The wounds left empty were recorded as negative control and the wounds treated with 0.1 g recombinant human epidermal growth factor gel (Pavay Gene pharmaceutical Co. Ltd) were recorded as positive control. All wounds were covered with a transparent dressing and bandages to protect the treatment. In order to delay the wounds to heal, a subcutaneous injection of Depomedrol (20 mg/kg BW) (Pfizer) was applied to all mice at day 0. At the determined time points, the mice were euthanized by cervical dislocation and the wounds were collected for histological analysis. The percentage of wound closure was calculated as:


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% of wound closure = 100×(original wound area-actual wound area)/original wound area, n=3. 2.9 Histological analysis and immunohistochemistry staining The wound tissues were fixed in 4% PFA for 24 h, embedded in paraffin and sectioned for further staining and analysis. The thickness of tissue sections was 8 µm. For histological analysis, the tissue sections were rehydrated and incubated with hematoxylin and eosin. For immunohistochemistry staining, the tissue sections were rehydrated and incubated with 0.01 M heated sodium citrate (Sinopharm Chemical Reagent Co., Ltd, 10019418) for antigen retrieval. 0.3 % H2O2/methanol (vol. /vol.) was used to permeate and inactivate, followed by blocking with 5% BSA (Sigma). After blocking, primary antibody solution containing rabbit anti-collagen I (diluted in PBS-1% BSA at 1:500) or rabbit anti-CD31 (diluted in PBS-1%BSA at 1:100) or mouse anti-α-SMA (diluted with PBS-1%BSA at 1:150) or mouse anti-human fibroblast surface protein (FSP) (diluted with PBS-1%BSA at 1:300) was used to incubate the samples at room temperature for 2 h. A GTVisionTM ΙΙΙ Detection System/Mo&Rb (Gene Tech (Shanghai) Co. Ltd, China) was used as secondary antibody and developer. The nuclei were stained with hematoxylin. After staining, all slices were observed with a microscope (Leica DM 2500, Germany) and images were taken with a CCD camera (Leica DFC 420C). The HDFs density was calculated by counting the cells migrated into wound site at 14 d after surgery in a microscope field with a magnification of 100X and the vessel density was calculated by counting the vessel numbers in a microscope field with magnification of 200X. Three fields were



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randomly chosen in each sample for being taken images. Totally 3 samples were taken images. 2.10 Statistical analysis The data were expressed as means ± standard deviation. Three independent experiments were carried out for validity and at least three samples per each 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. Statistical significance between two groups was performed with a Student’s t-test program and the differences were considered significant when p < 0.05 (*) or p < 0.01 (**).

3. Results 3.1 Effects of BG ionic dissolution products on HDF proliferation BG ionic dissolution products diluted at ratios ranging from 1/32 to 1/256 were used to study the effects of BG on HDF proliferation and cytotoxicity. It can be seen from Fig. 1A that the OD values in all groups are similar at each time point, which indicated that BG ionic dissolution products at 1/32 to 1/256 dilution ratios did not have much influence on HDF proliferation. Therefore, BG ionic dissolution products diluted at 1/64, 1/128 and 1/256 were used to culture HDFs for viability assessment, gene detection of growth factors and ECM components. Fig. 1B shows that BG ionic dissolution products diluted at 1/64, 1/128 and 1/256 could maintain the viability of HDFs as well as the control medium. 3.2 Expression of growth factors in fibroblasts in response to BG ionic dissolution


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products The effects of BG ionic dissolution products on the expression of bFGF, VEGF and EGF in fibroblast cell sheets are showed in Fig. 2 and different change patterns of these three growth factors in response to BG ionic dissolution products can be observed. Changes of bFGF expression in fibroblasts in response to BG ionic dissolution products show an irregular pattern. At 3 d, BG ionic dissolution products diluted at 1/64 stimulated bFGF expression in fibroblast cell sheets as compared to the control medium. However, the bFGF expression in fibroblast cell sheets cultured with control medium and BG 1/64 decreased at 7 d while BG 1/128 and BG 1/256 maintained the bFGF expression level in the fibroblasts. Therefore, at 7 d, bFGF expression in the fibroblasts cultured with BG ionic dissolution products diluted at 1/128 is highest among that in all fibroblasts (Fig. 2A). Regarding the effects of BG ionic dissolution products on VEGF expression, fibroblasts cultured with BG ionic dissolution products diluted at 1/128 always showed the highest expression of VEGF among all the fibroblasts cultured with different media, which is independent of the time points (Fig. 2B). Besides, VEGF expression in all fibroblasts increased with time. BG ionic dissolution products showed strong effects on EGF at 7 d. It seems that 3 days-culture is not enough for BG ionic dissolution products to stimulate EGF expression in fibroblasts since EGF expression in all fibroblasts cultured with different media is similar at 3 d. However, at 7 d, EGF expression decreased in fibroblasts cultured with control medium but increased in the fibroblasts cultured with all BG containing media, including BG 1/64, 1/128 and 1/256. Thus, as compared to



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the control medium, BG containing media stimulated EGF expression in fibroblasts cultured for 7 days. Particularly, group of fibroblasts cultured with BG ionic dissolution products diluted at 1/64 showed the highest EGF expression among all groups (Fig. 2C). These data were from three independent experiments to ensure the data validity. 3.3 Expression of collagen I, fibronectin and α-SMA in fibroblasts in response to BG ionic dissolution products The effects of BG ionic dissolution products on ECM composition and the differentiation of fibroblasts are presented in Fig. 3. The expression of collagen I, fibronectin and α-SMA in fibroblasts cultured with different media were first detected by Q-RT-PCR and the results are shown in Fig. 3 A, B, and C, respectively. After being cultured for 3 d, the fibroblasts cultured with BG ionic dissolution products diluted at 1/128 expressed higher collagen Ι than the control group and the highest fibronectin and α-SMA among all the groups. Expression of all these molecules in the fibroblasts cultured with different media was decreased at 7 d. In addition, at day 7, BG containing media did not stimulate collagen I production and α-SMA expression in fibroblasts as compared to the control medium except fibronectin. Since BG ionic dissolution products diluted with control medium at 1/128 played an important role in stimulating growth factors and ECM proteins, we used BG 1/128 to culture HDFs for 3 days for further immunofluorescence staining to reveal collagen I, fibronectin and α-SMA. Results were shown in Fig. 4 A. It can be seen that fibroblasts cultured with BG 1/128 produced more collagen I and fibronectin than


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those cultured in control medium. More α-SMA-positive staining could be observed when the fibroblast cell sheets were cultured with BG 1/128 as compared to the cells cultured with control medium. 3.4 Effects of BG ionic dissolution products on migration of fibroblasts BG ionic dissolution products diluted with control medium (without FBS) at 1/128 were used to detect the effects of BG on HDF migration and the results are shown in Fig. 4 B. At 0 h, the scratches with same width were made on bottom of each well covered with HDFs. After the cells were cultured with control medium (without FBS) and BG 1/128 for 24 h, the scratch in control group becomes slightly narrower while the scratch of BG containing group is almost disappeared, which indicated that the BG ionic dissolution products diluted at 1/128 stimulated the HDFs to migrate into the scratch area. Fig.4 C is the statistical analysis of HDF migration assay, the result showed that the scratch shrinkage percentage in BG containing medium group (91% ± 3.5%) was much larger than that in control group (37% ± 3%), and the result was statistical validity. 3.5 ICP results To understand the roles of main ions in the BG ionic dissolution products in affecting HDFs behaviors, ICP-AES was used to detect the ion concentration of BG ionic dissolution products diluted at different ratios with control medium as well as the ion concentration in control medium. The results were presented in Table 2. It shows that the concentrations of Ca and P ions in BG ionic dissolution products diluted at ratios ranging from 1/32 to 1/256 did not have much difference as compared



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with those in control medium. However, silicon ion concentration in BG containing medium, which is 2.63, 1.64, 0.88 and 0.65 µg/ml in BG ionic dissolution products diluted at ratios 1/32, 1/64, 1/128, 1/256, respectively, are much higher than that in control medium (0.35µg/ml). 3.6 Wound closure in response to cell sheets cultured with BG 1/128 and control medium Fig. 5 A shows the gross observation photos of wounds at day 3, 7 and 14 after being treated with different methods. The cell sheets cultured with BG 1/128 (BG cell sheet) and control medium (control cell sheet) showed different influences over wound closure at different time points, as confirmed by macroscopic analysis (Fig. 5 A) and closure measurements (Fig. 5 B). While for 3 days postoperative, the percentage of wound closure varied between different groups but BG cell sheet group shows the highest wound closure percentage among all groups. At day 7 after surgery, more wound closure was found in positive control group, control cell sheet group and BG cell sheet group as compared to that in negative control group and BG cell sheet group still maintained the highest wound closure percentage among all groups. At day 14, wounds treated by control cell sheets and BG cell sheets closed more than the wounds untreated (negative control) and treated by EGF gel (positive control). Wound treated with BG cell sheets closed a little more than those treated with control cell sheets (98% ± 2% vs 89% ± 8%). In conclusion, the wound closure percentage of BG cell sheet group is always highest in all groups at every time points. H&E staining of wound histology sections was shown in Fig. 6. At day 7, a


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neoepidermis underneath the eschar is observed in the control cell sheet group and BG cell sheet group while no neoepidermis is observed underneath the eschar of negative control group and positive control group. The granulation tissue functioned as a template for neodermis formation from day 7 onwards. The template is more organized for the groups in the positive control, control cell sheet and BG cell sheet at day 14 as compared to that in wound site at day 7. In addition, at day 14, neoepidermis formation can be observed in all the groups. However, the thickness of the neoepidermis is significantly different between all groups. The neoepidermis of BG cell sheet group is thickest in all groups although neoepidermis of control cell sheet group is thicker than those of the negative and positive control groups. 3.7 Human FSP staining in wound sites treated by different methods Results about human fibroblast surface protein staining in wound sites treated by control cell sheet and BG cell sheet at 7 d and 14 d were shown in Fig. 7 A in order to detect the migration of HDFs from fibroblast cell sheets into the wound sites. At 7 d, more HDFs can be seen in the wound bed treated by BG cell sheet than that treated by control cell sheet, which indicates that more HDFs migrated from BG cell sheet into wound bed than those migrated from control cell sheet. At 14 d, more HDFs showing positive staining of human fibroblast surface protein stayed on the surface of wound sites in control cell sheet group as compared to those on the surface of wound sites in BG cell sheet group. Meanwhile, less HDFs were observed in the wound bed treated by control cell sheet as compared to that in the wound bed treated by BG cell sheet. Fig. 7 B shows the statistical analysis result of the number of HDFs migrated into



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wound site at 14d after surgery. We counted the number of HDFs migrated into the wound site in a microscope field with a magnification of 100X. It can been seen that the average number of HDFs in the field in BG cell sheet group was about 24.5 ± 1.9, which was much more than that in control cell sheet group (15.3 ± 2.5) and it was statistical validity. 3.8 Deposition of collagen I in wound healing The results of immunohistochemistry staining for collagen I in wounds treated by different methods for 7 days are showed in Fig. 8. It shows that the staining intensity of collagen I in BG cell sheet group was strongest among all groups. Collagen I positive staining in control group is weakest among the four groups. Therefore, BG cell sheet group stimulated the deposition of collagen I in wound site. 3.9 Angiogenesis in wound healing Fig. 9 A shows the angiogenesis in wound site treated by different methods for 14 days, which was detected by CD 31 staining. It can be seen that the density of new vessels in wound site treated with cell sheets is much higher than that in wound site untreated or treated with EGF gel. However, as compared the control cell sheet with BG cell sheet, BG cell sheets significantly enhanced the blood vessel formation in wound site. It can be seen in Fig.9 B that statistical analysis showed a significant difference in the new blood vessel density in the four groups. The number of new vessels is about 10.6 ± 0.6 in a microscope field with the magnification of 200X in BG cell sheet group, which is the most in all groups. 3.10 α-SMA expression in wound sites treated by different methods


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The expression of α-SMA in wound sites treated by different methods for 14 day is presented in Fig. 10. Some α-SMA-positive staining can be seen in the cytoplasm of cells showing a spindled shape, which is a typical shape of myofibroblasts, indicating that these cells might be myofibroblasts differentiated from fibroblasts (arrows). In addition, some α-SMA-positive staining can be seen around new formed blood vessels (arrow heads). As smooth muscle cells, which also express α-SMA, dominate the middle layer of mature blood vessel, it can be reasoned that these cells may be smooth muscle cells. Amount of α-SMA-positive staining cells was different between different groups. Highest density of α-SMA-positive staining cells can be seen in BG cell sheet group among all groups. In addition, the α-SMA-positive staining cells in BG cell sheet group arranged much more compactly and orderly than those in other groups where α-SMA-positive staining cells randomly oriented. Furthermore, smooth muscle cells around the new blood vessels can be observed in almost all the groups. However, the number of the tubes is much more in BG cell sheet group which indicated that more mature blood vessels are formed in BG cell sheet group as compared to that in other groups.

4. Discussion It has been widely reported and accepted that biomaterials can affect cell behaviors in tissue engineering

1-4

. Since it has been reported that BG can stimulate

fibroblasts to secret growth factors and proteins that are beneficial for wound healing, in this study, we aim to construct a BG activated skin tissue engineering graft through combining BG and fibroblasts. The effects of BG on fibroblast behaviors and the



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wound healing ability of the bioactive BG-induced fibroblast cell sheet skin grafts have been investigated. Results confirmed that BG ionic dissolution products diluted at proper dilution ratios could stimulate fibroblast migration and secretion of growth factors, such as VEGF, bFGF and EGF, which could contribute wound healing. In addition, the production of proteins in fibroblast cell sheet ECM, such as collagen and fibronectin, was stimulated by BG. Due to the up-regulated growth factor secretion and protein enriched ECM, the effects of BG on wound healing were delivered to fibroblasts cell sheet skin graft that made it different from normal fibroblast cell sheet skin graft, thus the bioactive skin grafts possesses great bioactivity and significantly enhanced the wound healing. In the early phase of wound healing, the first two or three days after injury, fibroblasts mainly migrate and proliferate. It has been reported that stimulation of fibroblast migration can accelerate wound healing process 32-34. As fibroblasts migrate into wound site, they secrete growth factors contribute to wound healing and produce proteins in ECM that benefit wound healing. In addition, fibroblasts can differentiate into myofibroblasts to stimulate wound contraction, thus wound healing process can be accelerated

15, 18-19

. Furthermore, it has been proved that human cells can engraft

into wound sites and participate in the neotissue formation when the wounds are treated with human cell sheet

24, 35-36

. In this study, results showed that BG ionic

dissolution products significantly stimulated fibroblast migration in vitro. In vivo, we have confirmed that HDFs in cell sheet can migrate into the wound bed through human fibroblast surface protein staining. Once the migration speed of fibroblast in


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the cell sheets was accelerated by BG, more fibroblasts from the cell sheets migrated into the wound area as compared to the untreated fibroblast cell sheets. Then, these fibroblasts participated in and contributed to wound healing process. It has been widely accepted that angiogenesis was imperative for wound healing because the viability and activity of cells involved in wound healing process need oxygen and nutrients 2, 15, 37. In addition, the newly formed blood vessels are important part of granulation tissue which acts as a template for neodermis formation

38

.

However, angiogenesis speed induced by in vivo signals, which is normally several tenths of micrometers per day, is too slow to ensure adequate nutrient to the cells in the wound site

39

. Therefore, additional methods for promoting angiogenesis are

essential for wound healing. It had been reported that bFGF and VEGF are very critical growth factors for angiogenesis/vascularization in tissue regeneration, including wound healing 2, 40. bFGF had been used to treat wounds and results showed that the number of new capillaries in wound site was significantly increased after the wound was treated with bFGF for 5 days

41

. The critical roles of VEGF in

angiogenesis/vascularization have been widely studied and reported

2, 42-43

. As an

angiogenic growth factor, VEGF can induce endothelial cells to migrate into wound site, to proliferate and assemble into tubes. More specifically, VEGF increases the nitric oxide synthase and nitric oxide production in endothelial cells via the interactions with its receptors KDR and VEGF receptor-2 whose expression are also enhanced by VEGF 2, 44-45. In this study, in vitro results indicated that the expression of bFGF and VEGF in



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fibroblasts cultured with BG containing medium were up-regulated than those in fibroblasts cultured with control medium. From CD31 staining, we can see that more new blood vessels formed in the wound site treated with fibroblast cell sheets cultured with BG containing medium as compared to that in wound site treated with control cell sheet. Moreover, immunohistochemistry staining for α-SMA showed that smooth muscle cells could be observed around the new blood vessels, which also confirmed the formation of new blood vessels. It can be assumed that once fibroblast cell sheets treated with BG are applied to wound healing, the fibroblasts migrate into the wound bed, they will produce additional bFGF and VEGF to stimulate the formation of new blood vessels. Therefore, we propose that the up-regulation of bFGF and VEGF expression in human fibroblast cell sheet treated by BG ion extract may contribute to the formation of new blood vessels. Besides, in wound healing, chronic wound treatment is still a problem to be solved. Chronic wound is a wound that does not heal in a timely sequence which can be caused by many factors including vascular insufficiency, infection and pressure necrosis

46-47

. The primary stimulus for angiogenesis of granulation tissue is bFGF

and VEGF. For chronic wound, when these vascular growth factors are removed from the wounds, the angiogenesis in wound area is inhibited and granulation tissue is almost deficient in the wound, thus normal wound healing does not progress

46

. So,

once the angiogenesis is stimulated in chronic wound, there are abundant new capillaries to provide more nutrients and oxygen and to form more granulation tissue to accelerate chronic wound healing. In the current study, we delayed the wounds to


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heal through injecting Depomedrol into the mouse at day 0 and the results showed that the BG activated skin tissue engineering grafts combining BG and fibroblasts still can accelerate their healing process. Therefore, the BG activated skin tissue engineering grafts may be also beneficial for chronic wound healing in addition to helping acute wound healing. In our future studies, the effects of this BG activated skin tissue engineering grafts will be investigated on animal model with real chronic wounds, such as diabetic wounds. According to literatures, EGF can accelerate wound healing and has re-epithelialization ability

48-50

. It is reported that a wound dressing composed of

hyaluronic acid, collagen and EGF significantly promote re-epithelialization and decrease wound size as compared with the dressing without EGF

51

. In the present

study, BG ionic dissolution products enhanced the expression of EGF from fibroblasts in vitro. In addition, the neoepidermis formed in wound site treated by BG cell sheet group was thickest among all groups. Since EGF can accelerate re-epithelialization process in wound healing process, it can be reasoned that the up-regulation of EGF in fibroblast cell sheet by BG accelerated the neoepidermis formation. As angiogenic growth factors and epithelialization growth factor, VEGF, bFGF as well as EGF have been widely used for stimulating angiogenesis in tissue-engineered constructs and enhancing epithelialization, respectively

49, 52-54

Normally, these proteins are combined with biomaterials for controlling delivery

.

49

.

However, there are still some limitations for encapsulating these proteins by biomaterials as these proteins are easily to be denatured due to the unfavorable



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processing conditions and the short half-life of proteins 10. Besides, the in vivo release behavior of proteins is not easily controlled due to the complicated in vivo environment, which may induce risks

2,

10

. Recently, a biomaterial-based

proangiogenesis strategy has been proposed as studies demonstrated that some silicate bioactive materials, including BG, possess proangiogenic potential 10, 47. These studies showed that silicate bioactive materials could stimulate VEGF and/or bFGF expression from fibroblasts or endothelial cells, which subsequently initiate angiogenesis pathway

2, 55

. In this study, we confirmed that BG could stimulate not

only VEGF and bFGF but also EGF from fibroblasts, which may provide an alternative to the application of recombinant inductive growth factors. In addition, it has been thoroughly demonstrated that simultaneous delivery of multiple angiogenic factors is more effective than the delivery of a single angiogenic factor for enhancing vessel

density

and

maturity

56-57

.

Therefore,

another advantage

of

this

biomaterial-based proangiogenesis strategy is that the biomaterial can stimulate the target cells to express multiple types of growth factors simultaneously. Furthermore, the reported studies limited the application of silicate bioactive materials for stimulating vascularization in soft tissue engineering. Here, we may explore a potential application of BG in wound healing. Granulation tissue begins to appear in the wound site about two to five days after injury and it functioned as rudimental tissue. New blood vessels, fibroblasts, inflammatory cells, endothelial cells and a new provisional ECM compose the granulation tissue. The new provisional ECM is mainly composed of fibronectin and


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collagen produced by fibroblasts, which is different from the ECM of normal tissue 37. The collagen and fibronectin of new provisional ECM created a hydrated matrix, which facilitates cell migration, supports cell attachment and subsequent cell proliferation 58. Fibronectin acts as an extracellular scaffold for the cells migrated into wound site

59-60

. Repesh et al. reported that fibronectin was observed surrounding

fibroblasts. In addition, fibroblasts in granulation tissue were surrounded by more fibronectin than those at the wound periphery in rabbit61. Collagen matrix secreted by fibroblasts supports growth and differentiation of the cells involved in inflammation, angiogenesis and connective tissue construction

62-63

. Moreover, collagen deposition

increases the strength of the wound to provide resistance to the traumatic injury 64. In this study, in vitro results confirmed that the expression of fibronectin and collagen Ι was increased in the fibroblast skin graft cultured with BG containing medium. In addition, in vivo results showed that the collagen I deposition of the BG cell sheet group was much denser than that of the remaining groups. Therefore, after fibroblast skin graft cultured with BG ionic dissolution products were used in wound healing and the fibroblasts migrated into the wound bed, more collagen I were produced by those fibroblasts, which composes an enhanced new provisional ECM for cell attachment and cell proliferation as well as cell migration. Wound contraction occurs in the later phase of wound healing and late wound contraction can cause disfigurement and loss of functions

65

. In wound contraction,

myofibroblasts differentiated from fibroblasts are responsible for wound contraction 66

. The myofibroblasts are attracted by fibronectin and growth factors and move along



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the fibronectin in the ECM to reach the wound edges to contract the wounds 67. In this study, we demonstrated that BG ionic dissolution products stimulated the fibroblasts to differentiate into myofibroblasts, as confirmed by α-SMA immunofluorescence staining. In vivo, more α-SMA-positive cells were found in the BG cell sheet group as compared with that in the other groups, which indicates that BG-induced fibroblast skin graft could stimulate fibroblasts in the wound site to differentiate into myofibroblasts and these myofibroblasts may be responsible for wound contraction to accelerate wound healing. According to results in this study, we propose that one of the possible mechanisms for the BG activated skin tissue engineering grafts stimulating wound healing as following: First, the fibroblasts from the BG activated skin tissue engineering grafts were stimulated to migrate into wound site. As the BG also up-regulated the angiogenic growth factors expression and protein production in these fibroblasts, the angiogenesis, and granulation synthesis and epidermis formation in the wound site were then enhanced. Finally, the fibroblasts from the BG activated skin tissue engineering grafts were stimulated to differentiate into myofibroblasts, which accelerate wound contraction. The whole process is illustrated in Fig. 11. Interestingly, according to S. Zhao’s results, borate bioactive glass microfibers doped with 3.0 wt% CuO as wound dressings could promote endothelial cell migration, tubule formation and stimulate the expression of angiogenic growth factors in fibroblasts. When the wound dressings were used to treat skin defects, the wound dressings could accelerate wound healing. In addition, the angiogenesis and collagen deposition were stimulated.


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These results were quite similar with ours. It indicated that we could target the angiogenesis, collagen deposition or other related process in the wound healing to promote new ways and new materials to stimulate angiogenesis, collagen deposition or other aspects to stimulate wound healing68. In this study, BG ionic dissolution products were used to culture fibroblasts in order to obtain BG-induced fibroblast cell sheets with improved properties for wound healing. However, the types and concentrations of critical ions in the BG ionic dissolution products are important. The significant difference between the media containing BG ionic dissolution products and the control medium is the concentration of silicon ions. Silicon ions are very few in the control medium. Although BG ionic dissolution products diluted at ratios ranging from 1/32 to 1/256 did not have much influence on fibroblast proliferation and viability, BG ionic dissolution products diluted at 1/128 showed the strong stimulatory effects on the expression of growth factors and ECM proteins in fibroblasts. Therefore, 1/128 is the optimal dilution ratio for BG ionic dissolution products to improve the wound healing ability of fibroblast cell sheet. Concentration of silicon ion in BG ionic dissolution products diluted at 1/128 contains is about 0.88 µg/ml, which is in the range of effective silicon ion concentrations reported in our previous studies (0.7-1.8 µg/ml). Silicon ions with those concentrations have been reported to be effective for stimulating angiogenesis/vascularization2.

5. Conclusion In this study, we aimed to prepare a BG activated skin tissue engineering graft



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through combining BG and fibroblasts. We proved that BG ionic dissolution products could significantly activate fibroblasts through up-regulating the secretion of growth factors such as bFGF, VEGF, EGF in fibroblasts and increasing the production of collagen I, and fibronectin in the fibroblast ECM as well as stimulate the migration of fibroblasts. In vivo studies showed that the BG activated skin tissue engineering grafts could stimulate angiogenesis, enhance collagen deposition and accelerate wound contraction in the wound site, and thus accelerate wound healing process. Therefore, the BG activated skin tissue engineering grafts possess a great application potential in wound healing.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes No competing financial interests exist.

Acknowledgment This work was supported by Natural Science Foundation of China (Grant No.: 81190132, and 31470918), the Innovation Program of Shanghai Municipal Education Commission (Grant no. 14ZZ032) and the 2012 SMC-“Chenxing” Talent Program in Shanghai Jiaotong University.


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Figure 1. (A) Proliferation of HDFs cultured with different media, n=5. (B) The live-dead staining images of fibroblasts cultured with control medium and BG containing medium at dilution ratios from 1/64 to 1/256 for 3 days. Bar = 200 µm. Figure 2. (A) Gene expression of bFGF from HDF cultured with control medium and BG containing medium at dilution ratios 1/64, 1/128, 1/256. (B) Gene expression of VEGF from HDF cultured with control medium and BG containing medium at dilution ratios 1/64, 1/128, 1/256. (C) Gene expression of EGF from HDF cultured with control medium and BG containing medium at dilution ratios 1/64, 1/128, 1/256. *represents P

Bioglass Activated Skin Tissue Engineering ... - ACS Publications

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