Polydopamine-assisted PDGF-BB immobilization on PLGA fibrous substrate enhances wound healing via regulating anti-inflammatory and cytokine secretion

Xiao Yang; Peng Zhan; Xiuyan Wang; Qiushuang Zhang; Yi Zhang; Haitao Fan; Ranwei Li; Ming Zhang

doi:10.1371/journal.pone.0239366

. 2020 Sep 29;15(9):e0239366. doi: 10.1371/journal.pone.0239366

Polydopamine-assisted PDGF-BB immobilization on PLGA fibrous substrate enhances wound healing via regulating anti-inflammatory and cytokine secretion

Xiao Yang ¹, Peng Zhan ¹, Xiuyan Wang ¹, Qiushuang Zhang ¹, Yi Zhang ¹, Haitao Fan ^1,^*, Ranwei Li ^1,^*, Ming Zhang ^1,^*

Editor: Andrea Caporali²

PMCID: PMC7523965 PMID: 32991599

Abstract

Platelet-derived growth factor-bb (PDGF-BB) is a potent chemokine and mitogen for fibroblasts, keratinocytes, and vascular endothelium in the injured area, believed to be effective in wound healing. However, the short half-life of PDGF-BB and its rapid release from the wound surface limited its efficacy in vivo and vitro. To evaluate the wound healing effects of dorsal skin in SD rats with polydopamine-assisted immobilized PDGF-BB on PLGA nanofibrous substrate. First, the effects of pDA-coating and PDGF-BB immobilization on the morphology, compositions, and hydrophilicity of substrates were evaluated in details. Second, the wound healing effect of pDA/PLGA/PDGF-BB substrate was assessed in the dorsal skin of SD rats. Last, the cytokine analysis by ELISA method was employed to evaluate the advantages of pDA/PLGA/PDGF-BB substrate on anti-inflammatory, angiogenesis, and cellular proliferation. This method significantly improved the immobilization amount and stability of PDGF-BB on the substrate (p<0.01), further improved the hydrophilicity of substrates (p<0.05). Furthermore, the wound closure process was much more accelerated in the pDA/PLGA/PDGF-BB group (p<0.05). H&E and CD31 staining informed that the wound treated by pDA/PLGA/PDGF-BB substrate showed a high degree of regeneration and angiogenesis. The cytokine analysis showed that pDA significantly reduced the high level of inflammatory cytokines such as TNF-α (p<0.05). And the immobilized PDGF-BB significantly elevated the level of TGF-β and VEGF (p<0.05). The pDA/PLGA/PDGF-BB substrate showed great therapeutic effect on wound healing compared with other control groups via regulating anti-inflammatory, angiogenesis, and cellular proliferation. Absolutely, this report offered an available novel method for skin regeneration.

Introduction

Full-thickness skin injury caused by burns, bedsores or diabetic foot ulcers is an urgent clinical problem to be solved. In order to repair the damaged tissue including epidermis and dermis, various methods have been performed in wound area. Self-skin-grafting, often used as a conventional therapy, is considered to cause secondary injury and scarring. And it is not suitable for the extensive wounds beyond 2% of total body surface area due to the limited sources [1]. The application of heterogenous and allogeneic skin-grafting are supposed to solve these difficulties. However, it is hard to overcome the safety problems of biologics caused by immune rejection or cytotoxic responses [2–5]. Benefited from the development of the tissue engineering, human bioengineered skin implantations attracts a great interest in these field, and shows lots of advantages to defeat the problems mentioned above [1–7].

Poly[(rac-lactide)-co-glycolide] (PLGA), a FDA-approved polyester, has been widely used in the field of regenerative medicine due to its hydrolytic degradation and good controllability of macro properties. And electrospinning is a simple technique for the efficient production of micro to nano fibers from polymer solutions with high productivity and low cost. It was believed that electrospun fibers had the potential to achieve high drug loading and the ability to sustain drug release. Shih-Feng Chou studied the loading and release of tenofovir (TFV), a hydrophilic small molecule drug, into fibers of polycaprolactone (PCL) and poly (D,L-lactic-co-glycolic) acid (PLGA). The result showed that Sustained release of TFV depended on PLGA content. The PLGA fibers has outstanding sustained-release ability [8]. Amy Contreras’ group encapsulated a photosensitizer (PS) in PCL/PLGA fibers in the PS inert state, so that the antibacterial function would be activated on-demand via a visible light source [9]. This study successfully demonstrates the significant potential of PS-encapsulated electrospun fibers as photodynamically active biomaterial for antibiotic-free infection control. These studies fully demonstrated the advantages of PLGA electrospinning in drug sustained release.

A variety of positive factors such as antimicrobial materials [10–12], growth factors [13], and physical stimulation (electrical stimulation) [14] were used to functionalize the implants to achieve better therapeutic effect. Among these beneficial factors, growth factor has positive biological activity on the proliferation, migration and differentiation of cells, and plays an important role in skin regeneration. PDGF comprises a family of homo or heterodimeric growth factors including PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD. In the injury area, platelet-derived growth factor-bb (PDGF-BB) is a potent chemokine and mitogen for fibroblasts, keratinocytes, and vascular endothelium. It also stimulates macrophages to produce and secrete growth factors such as TGF-β. Hence, the PDGF-BB is often believed to be effective in wound healing. Recombinant human PDGF-BB (Becaplermin) is currently the only FDA-approved growth factor for the treatment of chronic trauma and has been successfully used for debridement and healing of advanced diabetic acromegaly ulcers based on gel dosage form [15]. Robert reported a novel therapy strategy combining PDGF-BB and a CXCR4 antagonist (AMD3100) to accelerate diabetic wound closure [16]. Specifically, it’s informed that the topical increase in PDGF-BB was beneficial to the homing/implantation of in situ progenitor cells into the wound. Not only in pharmacy, but also in biomaterial engineering, the PDGF-BB has been applied to develop novel skin graft. Xie's group created a bionic system that could be used as a scaffold to support wound healing, while simultaneously releasing VEGF and PDGF-BB in a way that mimicked different stages of angiogenesis and cell proliferation to promote wound healing [17]. A nanofibrous composite containing PDGF-BB-loaded nanoparticles was fabricated by electrospinning. Slow expelling of PDGF-BB improved the epithelium regeneration, collagen deposition, and functional tissue remodeling. With this design, an accelerated wound healing was achieved on a full thickness rat skin wound model. It was also informed that the PDGF-BB control releasing device was promising great potential in the treatment of skin regeneration.

However, the potency of PDGF-BB control releasing device was sometimes “uncontrollable” when applied in vitro and vivo due to the burst release in the wound site. Excessive local concentration of PDGF-BB may cause some unexpected side effects. In order to overcome these disadvantages, a growing interest was arisen in the development of biomaterials involving the immobilization of growth factors, which would allow these artificial materials to regulate specific cellular functions. In Jin’s report [18], epidermal growth factor (EGF) and other multiple epidermal induction factors were encapsulated in fibrous scaffold by core–shell electrospinning. No burst release was occurred. And an enhanced ability for ADSCs epidermal differentiation was achieved [13]. In a recent work, Wang fabricated a chitin film containing basic fibroblast growth factor with a chitin-binding domain as wound dressing. This system showed great advantages in uptake and release of bFGF, and finally determined to be a promising and effective strategy for wound dressing as it could induce vascularization in vivo [19]. However, nether encapsulating the growth factor into the material nor recombinant protein was a convenient method for immobilization of growth factor.

Recently, a pDA-assisted immobilization strategy simulating mussel-adhesion phenomena in nature was utilized to strengthen the incorporation of peptides or growth factors on implants. And the enhanced regenerative capacity of the modified implants was also positively verified in vitro and vivo. Under alkaline conditions, a poly(dopamine) layer is formed on materials through the catechol groups. And the dopamine may offer a lot of activated amino and phenyl to immobilize peptides and proteins through hydrogen-bond interaction. Jeong Seok Lee’s group fabricated a titanium (Ti) surface coated with pDA to facilitate the immobilization of BMP-2 [20]. The periodontal ligament stem cells (PDLSCs) cultured on pDA/BMP-2-Ti showed the highest osteogenic activity compared with those on the control Ti and pDA-coated Ti surfaces. Parivash Davoudi’s group immobilized Heparin and VEGF via self-polymerization and deposition of polydopamine (pDA) on polyurethane (PU) nanofibrous scaffolds [21]. The immobilized VEGF promoted the proliferation of human umbilical vein endothelial cells (HUVECs) through enhanced cells adhesion and cell-scaffold interactions. All these reports determined a great advantage of pDA-assisted growth factor immobilization on tissue regeneration.

The materials modified with different growth factors may get various bioactivities. So that exploring the specific bioactivity of each material modified with different growth factors is quite valuable and interesting. In this research, the pDA-assisted immobilization method was originally applied on bonding between PDGF-BB and PLGA fibrous substrate for in vivo wound healing. In this study, we chose the PLGA fibrous film fabricated by electrospinning as the substrate material. It was because that the PLGA has been proven to be a biocompatible and biodegradable material. And the porous mesh structure of nanofibrous substrate fabricated by electrospinning was beneficial for cell adhesion and migration. First, the effects of pDA-coating and PDGF-BB immobilization on the morphology, compositions, and hydrophilicity of substrates were evaluated in details. Second, the wound healing effect of pDA/PLGA/PDGF-BB substrate was assessed in the dorsal skin of SD rats. Last, the cytokine analysis by ELISA method was employed to evaluate the advantages of pDA/PLGA/PDGF-BB substrate on anti-inflammatory, angiogenesis, and cellular proliferation. The aim of this study was to establish a skin implantation with the potential to accelerate wound regeneration.

Materials and methods

Materials

PLGA (lactide/glycolide ratio = 75/25, MW = 100 000) was supplied by Changchun Institute of Applied Chemistry, Chinese Academy of Sciences (CIAC, China). Reagents were received from Sigma-Aldrich (St. Louis, USA) unless specified otherwise. The grade of reagents was analytical grade or higher. PDGF-BB was purchased from Peprotech (Trenton, USA).

Electrospinning of fibers

PLGA was dissolved in chloroform to obtain a 8% (w/v) solution. The polymer solutions were extruded from a 2.5 ml standard syringe attached to a 25G blunted stainless steel needle using a screw extruder at a flow rate of 1 mL/h. A high voltage of 15 kV was applied to drew the polymer solution into fibers on an aluminum foil wrapped collector at a distance of 10 cm from the needle tip. Fibers collected on foil were dried overnight under vacuum and used for the characterization and animal experiments.

PDA-assisted immobilization of PDGF-BB onto fibers

The pDA-coating process was carried out according to the previous report [22]. The PLGA fibers were soaked in dopamine solution (2 mg/mL in 10 mM Tris·HCl, pH 8.0) and shaken gently on an oscillator for 2 h under 37°C. In order to remove free dopamine, the pDA-coated fibers were adequately washed with deionized water five times. Afterward, PDGF-BB (100 ng/mL in PBS) was introduced on pDA/PLGA fibers for another 2 h under 4°C. The substrates were washed with deionized water three times to remove the free PDGF-BB. The PLGA without pDA coating was also set up to adsorb the PDGF-BB as contrast.

In order to demonstrated the stability of the polydopamine coating, a test was conducted. The PLGA and pDA/PLGA fibers were respectively soaked in 3 mL PBS solution (pH 7.2). 100 μL supernatant was collected as sample at each time point and detected the absorbance at 450 nm using an auto microplate reader (Tecan M200, Switzerland) as reported [23]. The quantitative absorption and release tests based on ELISA method were conducted as follow. When PDGF-BB was absorbed on to PLGA and pDA/PLGA fibers as described above, 100 μL supernatant was collected as sample at each time point. After incubation and wash, the fibers were directly soaked into 3 mL PBS (pH 7.2). 100 μL supernatant was collected as sample at each time point. All samples were diluted according to the range of ELISA kit., and detected quantificationally using the PDGF-BB DuoSet ELISA Development kit (R&D system). The absorption rate and release rate were calculated according to following formula:

A b s o r p t i o n r a t e (%) = (I n i t i a l A m o u n t - S a m p l e A m o u n t) / I n i t i a l A m o u n t \times 100 %

R e l e a s e r a t e (%) = S a m p l e A m o u n t / A b s o r p t i o n A m o u n t \times 100 %

Surface characterization

In order to prove the polydopamine coating on the fibers, the appearance photographs of PLGA and pDA/PLGA fibers were taken. The microstructure images of the membranes were obtained by an environmental scanning electron microscope (ESEM, XL30 ESEM-FEG, FEI). Fiber diameter for each sample was measured and recorded using ImageJ. The average fiber diameter was a statistic based on at least 5 photos from each sample. Fourier transform infrared spectroscopy (FT-IR, Perkin Elmer, FTIR-2000) was used to determine the chemical structure of the membranes. X-ray photoelectron spectroscopy (XPS) (Thermo) was employed to detect the protein-modified substrates. The elemental compositions of the modified substrates were obtained. The water contact angle was detected on a Kriss DSA 10 instrument. The shape of the water drop was recorded by a camera and the water contact angle was measured.

Animals and experimental protocol

All animal studies were conducted in accordance with the principles and procedures outlined in “Regulations for the Administration of Affairs Concerning Lab-oratory Animals”, approved by the National Council of China on October 31, 1988, and “The National Regulation of China for Care and Use of Laboratory Animals”, promulgated by the National Science and Technology Commission of China, on November 14, 1988 as Decree No. 2. Protocols were approved by the Committee of Jilin University Institutional Animal Care and Use. Healthy 8-week-old Sprague Dawley (SD) male rats (purchased from Changchun Institute of Biological Production) weighing ~200g were settled in Experimental Animal Center of Jilin University. Adequate supply of food and water was provided. The rats were randomly divided into four groups of four animals each, and anesthetized by i.p. injection of pentobarbital (40 mg/kg). The middle back area was shaved and disinfected with 75% alcohol. Then, two full-thickness orbicular wounds (diameter of 1.5 cm) were created on each side of their dorsum. Then, different substrates were cut into a suitable size and implanted onto the wounds, followed by injection of penicillin sodium for 4 days continuously. The wounds were covered with sterilized double-layer gauze. After surgery, the animals were placed in separate cages and nourished by assigned person. On day 7 postsurgery, the photographs of wounds were taken. The reduction in the size of wounds was determined from macroscopic images and by estimating the wound area using ImageJ software.

Immunohistochemistry and immunofluorescence

After 7 days, the rats were sacrificed with an i.p. injection of sodium pentobarbital. The skin scaffolds were excised and fixed in 4% formaldehyde for histological analysis. The fixed samples were embedded in paraffin. Paraffin sections were stained with hematoxylin and eosin (H&E, Sigma). For immunohistochemistry, the samples were stained with primary anti-rat CD31 antibody (1:500, Abcam), followed with HRP-labeled secondary antibody (1:2000, Abcam). After that the DAB was used as chromogenic substrate to locate the vessels. For immunofluorescence, the samples were stained with primary anti-rat CD31 antibody (1:500, Abcam), followed with Alexa-Fluor-488-labeled secondary antibody (1:2000, Abcam). Cell nucleus were dyed with 4,6-diamidino-2-phenylindole (DAPI) for 45 s. The graphs were taken and merged on an inverted microscope (Nikon E200).

Cytokine analysis

The samples were washed with PBS and smashed into powder in liquid nitrogen, extracted with 40mM Tris-HCl buffer solution (pH 8.0) containing 1 mM PMSF. The suspension was sonicated at 200W for 10 min for cell lysis and then centrifuged at 4℃, 12000 rpm for 10 min. The supernatant was collected and filtered by 0.22 μm membrane filtration. The cytokine level (TNF-α, VEGF, TGF-β) of the wound tissues was quantitatively detected by the ELISA kits (R&D, Shanghai).

Statistical analysis

All data would be reported in the form of mean±standard deviation. A statistical analysis was performed using ANOVA, followed by Tukey's Honestly Significant Difference (HSD) for multiple comparisons. The differences were considered statistically significant for p<0.05. Three or four independent replicates of each experiment were conducted.

Results and discussions

Surface characterization of modified fibers

As shown in S1 Fig, the pDA/PLGA fibers were dark brown in appearance, while the PLGA fibers were yellowish. It was due to the dark color of dopamine. In order to remove free dopamine, the pDA-coated fibers had been adequately washed with deionized water five times. Therefore, the dopamine was considered to be stability on the fibers. Even though a little of the dopamine would still fall off the materials.

The microtopography of the membranes was displayed on ESEM. According to the images (Fig 1)。 It was confirmed that the electrospun films were mainly composed by superposed fibers. The surface topography of the substrates was influenced by pDA coating, and a large amount of polymerized DA envelope was found on the pDA-coated or pDA-mediated peptide-immobilized substrates. As Wang’s report [22], pDA coating would obviously change the microtopography of the substrate. A large amount of polymerized DA particles was found on the pDA-coated substrate and increased the roughness of material surface. However, according to the result, there was no typical modified DA particles on PLGA fibers. The pDA was spread evenly on the material. It may be ascribed to the nHA in Wang’s material. The average fiber diameter for each sample was given in S1 Table. The morphology of fibers on the same sample might be non-uniform to a certain degree. It was because that many factors could influence the morphology of fibers. And the voltage during preparation was sometimes fluctuant slightly. However, there was no significant difference between samples.

FI-TR spectrum of pDA/PLGA/PDGF-BB, p-DA/PLGA, PLGA/PDGF-BB, and PLGA fibers was shown in Fig 2. The pDA/PLGA/PDGF-BB, pDA/PLGA, and PLGA/PDGF-BB nanofibrous films showed the characteristic amide I and amide II peaks of dopamine and PDGF-BB at 1596 cm-1 in addition to the spectrum of PLGA. Besides that, no significant exclusive peek was displayed.

The result of XPS in powerfully verified the results of FI-TR. Superposed peeks composed with the characteristic peeks were found at 398.3 eV, 399.8 eV and 402.1 eV, which respectively indicated amine (-NH-), imine (= N-),and (N+) in the spectrograms of pDA/PLGA/PDGF-BB, p-DA/PLGA, PLGA/PDGF-BB (Fig 3). Furthermore, it was found that the characteristic peeks on pDA/PLGA/PDGF-BB and p-DA/PLGA were much higher than that on PLGA/PDGF-BB. And the full width at half maximum (FWHM) of the characteristic peek on pDA/PLGA/PDGF-BB was slightly wider than that on p-DA/PLGA. Table 1 shows the elemental composition before and after peptide coating. The presence of nitrogen element (11.93%) indicated that pDA was successfully coated onto the PLGA nanofibrous films. Incorporation of PDGF-BB via pDA coating further increased the nitrogen content to 15.45%. The chemical deposition of the modified PLGA fibers from FT-IR and XPS analyses demonstrated the efficient immobilization of dopamine and PDGF-BB even after the extensive wash. The assisted PDGF-BB immobilization showed great adsorption advantage in both quantity and stability.

Fig 3 — XPS analyses demonstrated the efficient immobilization of PDGF-BB.

Table 1. Surface chemical composition of XPS.

(atomic concentration in %) of substrates analyzed by XPS, **p < 0.01.

	C	O	N
PLGA	65.28±0.95	29.73±0.98	0.11±0.03
PLGA/PDGF-BB	64.73±1.14	28.15±1.21	0.18±0.07
pDA/PLGA	62.12±1.67	24.64±1.24	11.23±0.43
pDA/PLGA/PDGF-BB	63.17±1.13	21.28±1.51	15.45±0.38^**

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Surface Chemical Composition (atomic concentration in %) of Substrates Analyzed by XPS,

**p < 0.01.

Fig 4 showed the hydrophilia performance of the nanofibrous films modified distinctively. The PLGA nanofibrous films was absolutely hydrophobic and the average contact angle was greater than 120°. In line with expectation, as the adhesion of different proteins, the hydrophilia of the membranes were more or less improved. However, the groups of PLGA/PDGF-BB was still hydrophobic, while the nanofibrous films of pDA/PLGA/PDGF-BB and pDA/PLGA performed as a high-hydrophilic material, indicating a significant increase of wettability due to the pDA coating on the substrates.

A polymeric structure with water contact angle less than 55° resulted in more cellular adhesion, enhanced proliferation rates, and improved cellular growth regardless of cell type. Parivash Davoudi’s study [21] indicated that the introduction of a large number of hydrophilic groups such as–OH,–COOH and–NH2 in the layer of polydopamine could effectively improve hydrophilicity of the PUNF scaffolds. In addition, immobilization of VEGF subsequently further decreased the water contact angle. Wang’s study [22] also proved that polymerized DA particles were noted on the PLGA film coated with pDA, and the surface wettability were increased. And the surface wettability was extremely important for cellular interaction with materials. A similar phenomenon was also observed in this study. Correspondingly, the wettability of pDA-coated substrates was extraordinarily improved, especially for the pDA/PLGA/PDGF-BB substrate.

Kinetics of absorption and release

The stability of the coating polydopamine was test. The result was shown in S2 Fig. The released dopamine will form nanoparticles in aqueous solution, turning the solution to brown. The color changing can be semiquantitatively detected under OD 450. According to the result, there is no significant difference between PLGA and pDA/PLGA fibers. It indicated that dopamine barely releases from the PLGA fibers. The release of drug is often accompanied by material degradation [24]. However, the situation was different in the polydopamine-assisted system. The coating of polydopamine on materials was formed by the covalent bonds and strong noncovalent forces, including hydrogen bonding, charge transfer, and π-stacking [25]. Polydopamine would usually undergo degradation in vitro in the presence of oxidizing agents such as H2O2. In vivo studies performed by Langer’s group have also demonstrated that It took 8 weeks for polydopamine implants to completely degrade [26]. It is quite a long time to wound healing. Therefore, the absorbed PDGF-BB most likely released from fibers due to rupture of hydrogen bond between PDGF-BB and polydopamine.

The kinetics of growth factor absorption and release on PLGA fibers were detected. As shown in Fig 5(A), At each indicated point, the absorption rate of PDGF-BB on pDA/PLGA was significantly higher than that on PLGA (p < 0.01), indicating that the retained PDGF-BB on pDA/PLGA was more than that on PLGA. In the in vitro release experiment [Fig 5(B)], sustained release of PDGF-BB on pDA/PLGA and PLGA was followed up to 120 mins. PDGF-BB quickly released from materials at the first 20 mins. After that, the PDGF-BB continued releasing slowly from PLGA fibers, whereas it could be sustained on pDA/PLGA from the 20 minutes to 120 minutes. During 120 minutes, the release rate of LBD-BDNF on on pDA/PLGA was significantly higher than that on PLGA (p < 0.01). Therefore, pDA/PLGA fibers could effectively absorb PDGF-BB, keep PDGF-BB retaining on and sustained releasing from substrate in vitro.

In vivo assessment of pDA/PLGA/PDGF-BB substrate

The wound healing effect of pDA/PLGA/PDGF-BB substrate was assessed in the dorsal skin of SD rats. To measure the wound size, as shown in Fig 6(A), the wound area was photographed every 2 days. The wound area measured each day was normalized by regarding the initial wound area as 100%. The reduction of wound area was calculated by extrapolating from the measured data [Fig 6(B)]. Although the wound size decreased by healing for the PLGA-treated control group, the wound closure process was more accelerated for groups treated with pDA/PLGA/PDGF-BB. The reduction of wound area for the case of pDA/PLGA/PDGF-BB conjugate was over 80%, which was greatly higher than 44.71% for the control group. Remarkably, it was also statistically higher than 55.28% of pDA/PLGA and 51.63% of PLGA/PDGF-BB, reflecting the therapeutic effect of PDGF-BB conjugates on skin wound healing. A combination therapy strategy raised by Robert [16] confirmed that the addition topical PDGF-BB is required to normalize marrow-derived progenitor cells homing/engraftment into the wound to accelerate the wound closure. An earlier research also informed that PDGF augments macrophage-mediated tissue debridement and granulation tissue formation [27]. The immobilization of PDGF prolonged the therapeutic effect, contributed in wound closure.

Histological analysis with H&E staining was performed for further therapeutic analysis (Fig 7). After 7 days, the damaged dermal tissue area was still large for the untreated. Actually, according to the results showed in Fig 6, after treatment with pDA/PLGA/PDGF-BB, the border between proliferative tissues and original tissues was ambiguous, while visible border could be observed in other groups. The borders in groups of PLGA/PDGF-BB and pDA/PLGA were not as clear as that in PLGA group. This result indicated that the PDGF-BB and pDA both could improve fusion between proliferative and original skin. Particularly, according to the result of CD31 staining (Fig 8), a lot of brown positive staining was observed in group of pDA/PLGA/PDGF-BB, which was believed to be neovascularization. PDA/PLGA/PDGF-BB substrate treated group showed a highly angiogenesis structure, which might be attributed to the facilitated penetration of PDGF-BB into deep dermal tissues and the prolonged residence of PDGF-BB conjugate. Most of the self-regeneration ability of skin was from basal layer [28, 29], hair follicle [29, 30] and dermis [31]. Therefore, the main task of repairing the full skin injury is to reconstruct the skin structure. It is recognized that extracellular matrix (ECM) deposition and vascularization are the most important in tissue regeneration including skin. PDGF has been accepted to stimulate the proliferation of various cells and the production of ECM [32]. In addition, the PDGF stimulates human fibroblasts to contract floating collagen matrices [33]. In the process of tissue remodeling, PDGF helps to adjust the mitotic activity or the productivity of collagen in dermal fibroblasts [34]. To vascularization, it is worth noting that the effect of PDGF is weaker than that of FGF and VEGF and is not essential for the initial formation of blood vessels [15]. But PDGF is particularly important in blood vessel maturation. The presence of the neovascularization indicated that the vessels were much more mature to take on more blood supply in group of pDA/PLGA/PDGF-BB. In vivo experiments have shown that PDGF is critical for the recruitment of pericytes, thereby increasing the structural integrity of blood vessels [32]. In this study, it was found that the immobilized PDGF-BB greatly enhanced ECM maturation and vascularization, and provide the necessary ECM and vascular structure for skin regeneration.

Fig 8 — Effects of the substitutes on neovascularization of wound area at day 7 after transplantation by examining the expression of CD31. (The bars = 50 μm).

Cytokine analysis

The cytokine analysis (Fig 9) in skin tissues also supported the therapeutic effect of pDA/PLGA/PDGF-BB substrate. The pDA/PLGA/PDGF-BB substrate reduced the high level of inflammatory cytokines such as TNF-α induced by wound generation more effectively than the groups of PLGA and PLGA/PDGF-BB (p<0.05). Interestingly, injury treated with pDA/PLGA/PDGF-BB showed the increased level of TNF-α than that in pDA/PLGA group. However, the difference was not significant (p>0.05). It was reported that the acidic degradation product of PLGA would lead inflammatory reaction in wound. And there was little research supporting the anti-inflammatory role of PDGF-BB in wound area. It was recognized that PDGF could stimulates chemotaxis of neutrophils and macrophages [15]. In other words, PDGF-BB preferred to increase the level of inflammatory cytokines rather than decrease it. The PDGF-BB-recruited immunocytes were available in anti-bacteria and removal of pathological tissue in the early stage of wound regeneration. It was beneficial to reduce the inflammatory reaction in late stage. So that the level of TNF-α in PLGA/PDGF-BB group was a little lower than that in PLGA group. However, the high level of TNF-α was still significantly reduced in pDA/PLGA/PDGF-BB and pDA/PLGA group. It is because that the pDA-coating improved biocompatibility of PLGA [22].

TGF-β could stimulate the cellular proliferation. And VEGF could induce the angiogenesis. In this work, the pDA/PLGA/PDGF-BB substrate also significantly elevated the level of TGF-β and VEGF (p<0.05), believed to benefit the skin tissue regeneration. A previous report has confirmed that PDGF could stimulate macrophages to produce and secrete growth factors such as TGF-β and VEGF [15]. Coincidentally, this phenomenon is verified in this study. Taken together, we could confirm the feasibility of pDA/PLGA/PDGF-BB substrate from the excellent therapeutic effect on skin wound healing due to the reduction of TNF-α and increasing of TGF-β and VEGF, which was related to the long-term release of the conjugated PDGF-BB.

Conclusion

In this research, we developed PDGF-BB conjugates incorporated into a PLGA fibers film via pDA-assisted immobilization for skin wound healing by facilitated delivery and prolonged residence of PDGF-BB in wound tissues. The pDA-mediated functionalization allowed for dependable and efficient immobilization of PDGF-BB onto the substrates. Then, the wound healing effect of pDA/PLGA/PDGF-BB substrate was assessed in the dorsal skin of SD rats. With the sustained function on the substrate, PDGF-BB efficiently penetrated into peripheral skin tissues around the wound area with a long residence time against the protease in the wound tissues. The pDA/PLGA/PDGF-BB substrate showed great therapeutic effect on wound healing compared with other control groups via regulating anti-inflammatory, angiogenesis, and cellular proliferation. Absolutely, this report offered an available novel method for skin regeneration.

Supporting information

S1 Fig. Appearance of materials.

The appearance photographs of (A) PLGA and (B)pDA/PLGA fibers.

(TIF)

Click here for additional data file.^{(3.5MB, tif)}

S2 Fig. Stability of coating polydopamine.

The releasing test of pDA from pDA/PLGA fibers. The PLGA fibers was set as control. All data would be reported in the form of mean±standard deviation.

(TIF)

Click here for additional data file.^{(4.4MB, tif)}

S3 Fig. Immunofluorescence staining of CD31.

Effects of the substitutes on neovascularization of wound area at day 7 after transplantation by examining the expression of CD31. (The bars = 50 μm).

(TIF)

Click here for additional data file.^{(7.4MB, tif)}

S1 Table. Statistic of fiber diameter of each sample.

Statistic of fiber diameter of each sample. All data would be reported in the form of mean±standard deviation.

(DOCX)

Click here for additional data file.^{(14.9KB, docx)}

S1 File

(RAR)

Click here for additional data file.^{(267.7KB, rar)}

Acknowledgments

We thank Changchun Institute of Applied Chemistry, Chinese Academy of Sciences for its supplement of materials during the research.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

M.Z. Program of Scientific Development of Jilin Province (20180101103JC) http://www.jlkjxm.com/. R.L. Program of Scientific Development of Jilin Province (20180101103JC) http://www.jlkjxm.com/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1.Bottcher-Haberzeth S, Biedermann T, Reichmann E. Tissue engineering of skin. Burns. 2010;36(4):450–60. 10.1016/j.burns.2009.08.016 PubMed PMID: WOS:000277856300002. [DOI] [PubMed] [Google Scholar]
2.Supp DM, Boyce ST. Engineered skin substitutes: practices and potentials. Clin Dermatol. 2005;23(4):403–12. 10.1016/j.clindermatol.2004.07.023 PubMed PMID: WOS:000231045600013. [DOI] [PubMed] [Google Scholar]
3.Mansbridge JN. Tissue-engineered skin substitutes in regenerative medicine. Curr Opin Biotech. 2009;20(5):563–7. 10.1016/j.copbio.2009.08.008 PubMed PMID: WOS:000272712000009. [DOI] [PubMed] [Google Scholar]
4.MacNeil S. Biomaterials for tissue engineering of skin. Mater Today. 2008;11(5):26–35. 10.1016/S1369-7021(08)70087-7 PubMed PMID: WOS:000255691600020. [DOI] [Google Scholar]
5.Dixit S, Baganizi DR, Sahu R, Dosunmu E, Chaudhari A, Vig K, et al. Immunological challenges associated with artificial skin grafts: available solutions and stem cells in future design of synthetic skin. J Biol Eng. 2017;11. doi: UNSP 49 10.1186/s13036-017-0089-9 PubMed PMID: WOS:000417878800001. [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Kamel RA, Ong JF, Eriksson E, Junker JPE, Caterson EJ. Tissue Engineering of Skin. J Am Coll Surgeons. 2013;217(3):533–55. 10.1016/j.jamcollsurg.2013.03.027 PubMed PMID: WOS:000324043300019. [DOI] [PubMed] [Google Scholar]
7.Wood FM. Skin regeneration: The complexities of translation into clinical practise. Int J Biochem Cell B. 2014;56:133–40. 10.1016/j.biocel.2014.10.025 PubMed PMID: WOS:000347127400016. [DOI] [PubMed] [Google Scholar]
8.Chou SF, Woodrow KA. Relationships between mechanical properties and drug release from electrospun fibers of PCL and PLGA blends. J Mech Behav Biomed. 2017;65:724–33. 10.1016/j.jmbbm.2016.09.004 PubMed PMID: WOS:000390625100067. [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Amy Contreras MJR, Wood Simon, Jessica D. Schiffman, Tronci Giuseppe. Photodynamically Active Electrospun Fibers for Antibiotic-Free Infection Control. ACS Appl Bio Mater. 2019;2:4258–70. 10.1021/acsabm.9b00543 [DOI] [PubMed] [Google Scholar]
10.Schneider RK, Anraths J, Kramann R, Bornemann J, Bovi M, Knuchel R, et al. The role of biomaterials in the direction of mesenchymal stem cell properties and extracellular matrix remodelling in dermal tissue engineering. Biomaterials. 2010;31(31):7948–59. 10.1016/j.biomaterials.2010.07.003 PubMed PMID: WOS:000282109100009. [DOI] [PubMed] [Google Scholar]
11.Zhou T, Sui BY, Mo XM, Sun J. Multifunctional and biomimetic fish collagen/bioactive glass nanofibers: fabrication, antibacterial activity and inducing skin regeneration in vitro and in vivo. Int J Nanomed. 2017;12:3495–507. 10.2147/Ijn.S132459 PubMed PMID: WOS:000401001900002. [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Song JF, Zhang PF, Cheng L, Liao Y, Xu B, Bao R, et al. Nano-silver in situ hybridized collagen scaffolds for regeneration of infected full-thickness burn skin. J Mater Chem B. 2015;3(20):4231–41. PubMed PMID: WOS:000354450300013. 10.1039/c5tb00205b [DOI] [PubMed] [Google Scholar]
13.Jin GR, Prabhakaran MP, Kai D, Ramakrishna S. Controlled release of multiple epidermal induction factors through core-shell nanofibers for skin regeneration. Eur J Pharm Biopharm. 2013;85(3):689–98. 10.1016/j.ejpb.2013.06.002 PubMed PMID: WOS:000330200400043. [DOI] [PubMed] [Google Scholar]
14.Fleischli JG, Laughlin TJ. Electrical stimulation in wound healing. The Journal of foot and ankle surgery: official publication of the American College of Foot and Ankle Surgeons. 1997;36(6):457–61. 10.1016/s1067-2516(97)80099-3 . [DOI] [PubMed] [Google Scholar]
15.Barrientos S, Stojadinovic O, Golinko MS, Brem H, Tomic-Canic M. Growth factors and cytokines in wound healing. Wound repair and regeneration: official publication of the Wound Healing Society [and] the European Tissue Repair Society. 2008;16(5):585–601. 10.1111/j.1524-475X.2008.00410.x . [DOI] [PubMed] [Google Scholar]
16.Allen RJ Jr., Soares MA, Haberman ID, Szpalski C, Schachar J, Lin CD, et al. Combination therapy accelerates diabetic wound closure. PloS one. 2014;9(3):e92667 10.1371/journal.pone.0092667 [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Xie Z, Paras CB, Weng H, Punnakitikashem P, Su LC, Vu K, et al. Dual growth factor releasing multi-functional nanofibers for wound healing. Acta Biomater. 2013;9(12):9351–9. 10.1016/j.actbio.2013.07.030 [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Park JW, Hwang SR, Yoon IS. Advanced Growth Factor Delivery Systems in Wound Management and Skin Regeneration. Molecules. 2017;22(8). 10.3390/molecules22081259 [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Wang Y, Fu C, Wu Z, Chen L, Chen X, Wei Y, et al. A chitin film containing basic fibroblast growth factor with a chitin-binding domain as wound dressings. Carbohydrate polymers. 2017;174:723–30. 10.1016/j.carbpol.2017.05.087 . [DOI] [PubMed] [Google Scholar]
20.Lee JS, Lee JC, Heo JS. Polydopamine-assisted BMP-2 immobilization on titanium surface enhances the osteogenic potential of periodontal ligament stem cells via integrin-mediated cell-matrix adhesion. Journal of cell communication and signaling. 2018;12(4):661–72. 10.1007/s12079-018-0468-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Davoudi P, Assadpour S, Derakhshan MA, Ai J, Solouk A, Ghanbari H. Biomimetic modification of polyurethane-based nanofibrous vascular grafts: A promising approach towards stable endothelial lining. Materials science & engineering C, Materials for biological applications. 2017;80:213–21. 10.1016/j.msec.2017.05.140 . [DOI] [PubMed] [Google Scholar]
22.Wang Z, Chen L, Wang Y, Chen X, Zhang P. Improved Cell Adhesion and Osteogenesis of op-HA/PLGA Composite by Poly(dopamine)-Assisted Immobilization of Collagen Mimetic Peptide and Osteogenic Growth Peptide. ACS applied materials & interfaces. 2016;8(40):26559–69. 10.1021/acsami.6b08733 . [DOI] [PubMed] [Google Scholar]
23.Zhang C, Miyatake H, Wang Y, Inaba T, Wang Y, Zhang P, et al. A Bioorthogonal Approach for the Preparation of a Titanium-Binding Insulin-like Growth-Factor-1 Derivative by Using Tyrosinase. Angewandte Chemie. 2016;55(38):11447–51. 10.1002/anie.201603155 . [DOI] [PubMed] [Google Scholar]
24.Tronci G, Yin J, Holmes RA, Liang H, Russell SJ, Wood DJ. Protease-sensitive atelocollagen hydrogels promote healing in a diabetic wound model. J Mater Chem B. 2016;4(45):7249–58. 10.1039/c6tb02268e PubMed PMID: WOS:000388894600007. [DOI] [PubMed] [Google Scholar]
25.Liu YL, Ai KL, Lu LH. Polydopamine and Its Derivative Materials: Synthesis and Promising Applications in Energy, Environmental, and Biomedical Fields. Chem Rev. 2014;114(9):5057–115. 10.1021/cr400407a PubMed PMID: WOS:000336078200013. [DOI] [PubMed] [Google Scholar]
26.Bettinger CJ, Bruggeman PP, Misra A, Borenstein JT, Langer R. Biocompatibility of biodegradable semiconducting melanin films for nerve tissue engineering. Biomaterials. 2009;30(17):3050–7. 10.1016/j.biomaterials.2009.02.018 PubMed PMID: WOS:000266194900003. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Lederle W, Stark HJ, Skobe M, Fusenig NE, Mueller MM. Platelet-derived growth factor-BB controls epithelial tumor phenotype by differential growth factor regulation in stromal cells. The American journal of pathology. 2006;169(5):1767–83. 10.2353/ajpath.2006.060120 [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Fuchs E. Skin stem cells: rising to the surface. The Journal of cell biology. 2008;180(2):273–84. 10.1083/jcb.200708185 [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Blanpain C, Fuchs E. Epidermal stem cells of the skin. Annu Rev Cell Dev Bi. 2006;22:339–73. 10.1146/annurev.cellbio.22.010305.104357 PubMed PMID: WOS:000242325100015. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Brockmann I, Ehrenpfordt J, Sturmheit T, Brandenburger M, Kruse C, Zille M, et al. Skin-Derived Stem Cells for Wound Treatment Using Cultured Epidermal Autografts: Clinical Applications and Challenges. Stem Cells Int. 2018. doi: Artn 4623615 10.1155/2018/4623615. PubMed PMID: WOS:000429623300001. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Lorenz K, Sicker M, Schmelzer E, Rupf T, Salvetter J, Schulz-Siegmund M, et al. Multilineage differentiation potential of human dermal skin-derived fibroblasts. Exp Dermatol. 2008;17(11):925–32. 10.1111/j.1600-0625.2008.00724.x PubMed PMID: WOS:000259936100004. [DOI] [PubMed] [Google Scholar]
32.Lin H, Chen B, Sun W, Zhao W, Zhao Y, Dai J. The effect of collagen-targeting platelet-derived growth factor on cellularization and vascularization of collagen scaffolds. Biomaterials. 2006;27(33):5708–14. 10.1016/j.biomaterials.2006.07.023 . [DOI] [PubMed] [Google Scholar]
33.Rhee S, Grinnell F. P21-activated kinase 1: convergence point in PDGF- and LPA-stimulated collagen matrix contraction by human fibroblasts. The Journal of cell biology. 2006;172(3):423–32. 10.1083/jcb.200505175 [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Jinnin M, Ihn H, Mimura Y, Asano Y, Yamane K, Tamaki K. Regulation of fibrogenic/fibrolytic genes by platelet-derived growth factor C, a novel growth factor, in human dermal fibroblasts. Journal of cellular physiology. 2005;202(2):510–7. 10.1002/jcp.20154 . [DOI] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0239366.r001

Decision Letter 0

Andrea Caporali

15 Apr 2020

PONE-D-19-33330

Polydopamine-assisted PDGF-BB immobilization on PLGA nanofibrous substrate enhances wound healing via regulating anti-inflammatory and cytokine secretion

PLOS ONE

Dear Dr Zhang,

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Reviewer #2: No

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Reviewer #1: It seems a real control untreated group of animals is lacking; Can you explain why?

Figure 6: authors claim that the arrows indicate the border between regenerated tissue and normal tissues. Although this is not clearly visible, wound healing at 7 days should be in the proliferative phase and not in the absence of "regenerated" tissue. "Regenerated" is not a term that can be used in skin wound healing: skin repairs, does not regenerate. Can you clarify?

Figure 7: blood vessels are not visible. The antibody seems to be unspecific, please provide high magnification where endothelial cells are stained.

Reviewer #2: The authors investigate the immobilisation of Platelet-derived growth factor-bb on the surface of PLGA electrospun fibres, aiming at controlled growth factor delivery to stimulate wound healing in full thickness wounds in rats. To deliver on this, a polydopamine coating is attempted on the fibres. My main concerns are around the material design and characterisation, specifically:

1) The data presented do not fully support the formation of a polydopamine coating on the fibres; can the authors carry out some degradation experiments to check that no dopamine leaches out from the fibres? At the moment, the data supports that there is deposition of dopamine species, yet the formation of the polydopamine coating is not fully demonstrated. The authors should provide convincing evidence for this claim.

2) What is the mechanism of growth factor immobilisation and what is the kinetics of growth factor release? The authors should provide data on this using controls made doped with the growth factor in the absence of the dopamine treatment. These data would also help in explaining some of the results obtained with the study in vivo.

3) The SEM images show the internal structure of the fibres. The Authors should assess the average fibre diameter for each sample in order to provide quantitative results. Why do the fibres of the growth factor-loaded scaffolds following dopamine treatment look thicker than all the other sample groups?

4) The authors attribute the increased wound closure of the wounds in receipt of the growth factor loaded and dopamine treated scaffolds to the prolonged PDGF therapeutic effect, due to the effect of the immobilisation. How is this immobilisation achieved and can the authors support this with release data in vitro?

The manuscript also lacks a clear motivation as to why this study is original. The authors state in the introduction that growth factor immobilisation has already been reported. What is the rational for this study then?

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PLoS One. 2020 Sep 29;15(9):e0239366. doi: 10.1371/journal.pone.0239366.r002

Author response to Decision Letter 0

16 May 2020

First of all, we would like to express our great appreciation to the reviewers for their helpful comments and suggestions.

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

Response: Thank you for your kind comments. The manuscript has been revised to meet PLOS ONE's style requirements.

Response: Thank you for your kind comments.

Page 9-10：……All animal studies were conducted in accordance with the principles and procedures outlined in “Regulations for the Administration of Affairs Concerning Lab-oratory Animals”, approved by the National Council of China on October 31, 1988, and “The National Regulation of China for Care and Use of Laboratory Animals”, promulgated by the National Science and Technology Commission of China, on November 14, 1988 as Decree No. 2. Protocols were approved by the Committee of Jilin University Institutional Animal Care and Use. Healthy 8-week-old Sprague Dawley (SD) male rats (purchased from Changchun Institute of Biological Production) weighing ~200g were settled in Experimental Animal Center of Jilin University. Adequate supply of food and water was provided. The rats were randomly divided into four groups of four animals each, and anesthetized by i.p. injection of pentobarbital (40 mg/kg). The middle back area was shaved and disinfected with 75% alcohol. Then, two full-thickness orbicular wounds (diameter of 1.5 cm) were created on each side of their dorsum. Then, different substrates were cut into a suitable size and implanted onto the wounds, followed by injection of penicillin sodium for 4 days continuously. The wounds were covered with sterilized double-layer gauze. After surgery, the animals were placed in separate cages and nourished by assigned person. On day 7 postsurgery, the photographs of wounds were taken. The reduction in the size of wounds was determined from macroscopic images and by estimating the wound area using ImageJ software. ……

Reviewer #1:

1. It seems a real control untreated group of animals is lacking; Can you explain why?

Response: Thank you for your kind comments. The regeneration materials based on PLGA nanofibrous substrate has been widely reported. However, the lack of bioactivity was still a burning problem. This study focused on the enhanced bioactivity of PLGA substrate profited from the polydopamine-assisted PDGF-BB immobilization. An untreated control group was surely useful to show the natural repair of wound. Nevertheless, in order to extrude this emphasis, the PLGA group was set up as control. The results also proved that the ability of PLGA substrate on skin repair was significantly improved by polydopamine-assisted PDGF-BB immobilization.

2.Figure 6: authors claim that the arrows indicate the border between regenerated tissue and normal tissues. Although this is not clearly visible, wound healing at 7 days should be in the proliferative phase and not in the absence of "regenerated" tissue. "Regenerated" is not a term that can be used in skin wound healing: skin repairs, does not regenerate. Can you clarify?

Response: Thank you for your kind comments. In this part, the main task was to point out the positive effect of polydopamine-assisted PDGF-BB immobilization on fusion between newborn and original skin. Actually, according to the results showed in Figure 6, after treatment with pDA/PLGA/PDGF-BB, the border between proliferative tissues and original tissues was ambiguous, while visible border could be observed in other groups. The borders in groups of PLGA/PDGF-BB and pDA/PLGA were not as clear as that in PLGA group. This result indicated that the PDGF-BB and pDA both could improve fusion between proliferative and original skin. Detailed description of these results was added in the manuscript. However, your comments made me realize that the description was incorrect. Thank you for your correction and we have revised it in the manuscript according to the comments.

Page 13: “……After 7 days, the damaged dermal tissue area was still large for the untreated. Actually, according to the results showed in Figure 6, after treatment with pDA/PLGA/PDGF-BB, the border between proliferative tissues and original tissues was ambiguous, while visible border could be observed in other groups. The borders in groups of PLGA/PDGF-BB and pDA/PLGA were not as clear as that in PLGA group. This result indicated that the PDGF-BB and pDA both could improve fusion between proliferative and original skin. Particularly, according to……”

3.Figure 7: blood vessels are not visible. The antibody seems to be unspecific, please provide high magnification where endothelial cells are stained.

Response: Thank you for your kind comments. Anti-CD31 antibody is commonly used in most histopathology laboratories as a vascular marker and is thus readily available to most histopathologists. During routine practice it became apparent that CD31 was also a good marker of microthrombus in tissue sections. And the microthrombus was quite common in post-mortem tissue and located in some mature vessels. In Figure 7, a lot of granulated positive staining was observed in group of pDA/PLGA/PDGF-BB, which was believed to be microthrombus. These microthrombi were located in the lumen of staining-positive cells. Moreover, the presence of the microthrombi indicated that the vessels were mature enough to take on more blood supply. In order to make this part more clear, the images were updated. And the neovessels were point out with arrows in merged images. In addition, the manuscript was also revised.

Page 13: ……Particularly, according to the result of CD31 staining (Figure 7), a lot of granulated positive staining was observed in group of pDA/PLGA/PDGF-BB, which was believed to be microthrombus. These microthrombi were located in the lumen of staining-positive cells. PDA/PLGA/PDGF-BB substrate treated group showed a highly angiogenesis structure, which might be attributed to the facilitated penetration of PDGF-BB into deep dermal tissues and the prolonged residence of PDGF-BB conjugate……

Page 14：……But PDGF is particularly important in blood vessel maturation. During routine practice it became apparent that CD31 was also a good marker of microthrombus in tissue sections. And the microthrombus was quite common in post-mortem tissue and located in some mature vessels.(29) The presence of the microthrombi indicated that the vessels were much more mature to take on more blood supply in group of pDA/PLGA/PDGF-BB. In vivo experiments have shown that PDGF is critical for the recruitment……

Reviewer #2:

The authors investigate the immobilization of Platelet-derived growth factor-bb on the surface of PLGA electrospun fibers, aiming at controlled growth factor delivery to stimulate wound healing in full thickness wounds in rats. To deliver on this, a polydopamine coating is attempted on the fibers. My main concerns are around the material design and characterization, specifically:

1) The data presented do not fully support the formation of a polydopamine coating on the fibers; can the authors carry out some degradation experiments to check that no dopamine leaches out from the fibers? At the moment, the data supports that there is deposition of dopamine species, yet the formation of the polydopamine coating is not fully demonstrated. The authors should provide convincing evidence for this claim.

Response:

Thank you for your kind comments. In order to prove the polydopamine coating on the fibers, the appearance photographs of PLGA and pDA/PLGA fibers were taken. As shown in Figure S1, the pDA/PLGA fibers were dark brown in appearance, while the PLGA fibers were yellowish. It was due to the dark color of dopamine. In order to remove free dopamine, the pDA-coated nanofibers had been adequately washed with deionized water five times. Therefore, the dopamine was considered to be stability on the fibers. Even though a little of the dopamine would still fall off the materials.

Page 8: ……In order to prove the polydopamine coating on the fibers, the appearance photographs of PLGA and pDA/PLGA fibers were taken. The microstructure images of the membranes were obtained by an environmental scanning electron microscope (ESEM, XL30 ESEM-FEG, FEI).……

Page 10：……As shown in Figure S1, the pDA/PLGA fibers were dark brown in appearance, while the PLGA fibers were yellowish. It was due to the dark color of dopamine. In order to remove free dopamine, the pDA-coated nanofibers had been adequately washed with deionized water five times. Therefore, the dopamine was considered to be stability on the fibers. Even though a little of the dopamine would still fall off the materials.……

2) What is the mechanism of growth factor immobilization and what is the kinetics of growth factor release? The authors should provide data on this using controls made doped with the growth factor in the absence of the dopamine treatment. These data would also help in explaining some of the results obtained with the study in vivo.

Response: Thank you for your kind comments. the mechanism of growth factor immobilization has been clarified in the part of introduction. Under alkaline conditions, a poly(dopamine) layer is formed on materials through the catechol groups. And the dopamine may offer a lot of activated amino and phenyl to immobilize peptides and proteins through hydrogen-bond interaction.

In order to clarify the kinetics of growth factor absorption and release on PLGA fibers, the quantitative absorption and release tests based on ELISA method were conducted. Related content was added in revised manuscript.

Page 8:……The substrates were washed with deionized water three times to remove the free PDGF-BB. The PLGA without pDA coating was also set up to adsorb the PDGF-BB as contrast.

The quantitative absorption and release tests based on ELISA method were conducted as follow. When PDGF-BB was absorbed on to PLGA and pDA/PLGA fibers as described above, 100 μL supernatant was collected as sample at each time point. After incubation and wash, the fibers were directly soaked into 3 mL PBS (pH 7.2). 100 μL supernatant was collected as sample at each time point. All samples were diluted according to the range of ELISA kit., and detected quantificationally using the PDGF-BB DuoSet ELISA Development kit (R&D system). The absorption rate and release rate were calculated according to following formula:

Absorption rate (%) = (Initial Amount-Sample Amount)/Initial Amount × 100%

Release rate (%) = Sample Amount/ Absorption Amount × 100%

……

Page 13: ……

3.2 Kinetics of growth factor absorption and release on PLGA fibers

The kinetics of growth factor absorption and release on PLGA fibers were detected. As shown in Figure 5(A), At each indicated point, the absorption rate of PDGF-BB on pDA/PLGA was significantly higher than that on PLGA (p < 0.01), indicating that the retained PDGF-BB on pDA/PLGA was more than that on PLGA. In the in vitro release experiment [Figure 5(B)], sustained release of PDGF-BB on pDA/PLGA and PLGA was followed up to 120 mins. PDGF-BB quickly released from materials at the first 20 mins. After that, the PDGF-BB continued releasing slowly from PLGA fibers, whereas it could be sustained on pDA/PLGA from the 20 minutes to 120 minutes. During 120 minutes, the release rate of LBD-BDNF on on pDA/PLGA was significantly higher than that on PLGA (p < 0.01). Therefore, pDA/PLGA fibers could effectively absorb PDGF-BB, keep PDGF-BB retaining on and sustained releasing from substrate in vitro.……

3) The SEM images show the internal structure of the fibers. The Authors should assess the average fiber diameter for each sample in order to provide quantitative results. Why do the fibers of the growth factor-loaded scaffolds following dopamine treatment look thicker than all the other sample groups?

Response: Thank you for your kind comments. In the supporting information of revised manuscript, a table containing the average fiber diameter for each sample was given (Table S1.). The result was a statistic based on at least 5 photos from each sample. However, there was no significant difference between samples. Many factors could influence the morphology of fibers. And the voltage during preparation was sometimes fluctuant slightly. So that the morphology of fibers on the same sample might be non-uniform to a certain degree. The SEM images shown in the original manuscript might be misleading. The image of the growth factor-loaded scaffolds following dopamine treatment has been replaced by another image of the same sample.

Page 8: ……The microstructure images of the membranes were obtained by an environmental scanning electron microscope (ESEM, XL30 ESEM-FEG, FEI). Fiber diameter for each sample was measured and recorded using ImageJ. The average fiber diameter was a statistic based on at least 5 photos from each sample. Fourier transform infrared spectroscopy (FT-IR, Perkin Elmer, FTIR-2000) was used to determine the chemical structure of the membranes.……

Page 11:……It may be ascribed to the nHA in Wang’s material. The average fiber diameter for each sample was given in Table S1. The morphology of fibers on the same sample might be non-uniform to a certain degree. It was because that many factors could influence the morphology of fibers. And the voltage during preparation was sometimes fluctuant slightly. However, there was no significant difference between samples. ……

S1 Table. Statistic of fiber diameter of each sample. All data would be reported in the form of mean±standard deviation.

PLGA PLGA/PDGF-BB pDA/PLGA pDA/PLGA/PDGF-BB

Fiber diameter (μm) 1.53±0.64 1.51±0.71 1.61±0.68 1.57±0.74

4) The authors attribute the increased wound closure of the wounds in receipt of the growth factor loaded and dopamine treated scaffolds to the prolonged PDGF therapeutic effect, due to the effect of the immobilization. How is this immobilization achieved and can the authors support this with release data in vitro?

Response: Thank you for your kind comments. the mechanism of growth factor immobilization and the release data in vitro has been given under the comment 2. I hope that the response was clear to answer the questions.

5）The manuscript also lacks a clear motivation as to why this study is original. The authors state in the introduction that growth factor immobilization has already been reported. What is the rational for this study then?

Response: Thank you for your kind comments. As introduced in manuscript, the Polydopamine-assisted growth factor immobilization has been used in many sections of tissue engineering. The materials modified with different growth factors may get various bioactivities. So that exploring the specific bioactivity of each material modified with different growth factors is quite valuable and interesting. In this research, the pDA-assisted immobilization method was originally applied on bonding between PDGF-BB and PLGA fibrous substrate for in vivo wound healing. Furthermore, this report instructively confirmed the reduction of TNF-α and increasing of TGF-β and VEGF caused by the long-term effect of the conjugated PDGF-BB on pDA/PLGA/PDGF-BB substrate. These results would be helpful to illuminate the underlying mechanism in subsequent studies.

Page 6: ……The materials modified with different growth factors may get various bioactivities. So that exploring the specific bioactivity of each material modified with different growth factors is quite valuable and interesting. In this research, the pDA-assisted immobilization method was originally applied on bonding between PDGF-BB and PLGA fibrous substrate for in vivo wound healing. In this study, we chose the PLGA……

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PLoS One. doi: 10.1371/journal.pone.0239366.r003

Decision Letter 1

Andrea Caporali

11 Jun 2020

PONE-D-19-33330R1

Polydopamine-assisted PDGF-BB immobilization on PLGA fibrous substrate enhances wound healing via regulating anti-inflammatory and cytokine secretion

PLOS ONE

Dear Dr. Zhang,

Specifically, the question on the stability of the coating is partially answered and it would need an additional degradation test to check the kinetics of the dopamine released. Moreover, CD31 immunohistochemistry in Figure 7 should improved.

Please submit your revised manuscript by Jul 26 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

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Andrea Caporali, PhD

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: No

**********

6. Review Comments to the Author

Reviewer #1: Dear authors,

thank you for the provided responses. However it is my opinon that green staining that is seen in figure 7 depends on red blood cell unspecific fuorescence.

without high magnification images I cannot say that this is acceptable.

Reviewer #2: The authors have clarified the points raised previously with additional results that support their claims. The question on the stability of the coating is still only partially answered and it would need an additional degradation test to check the kinetics of any dopamine released. I would invite the authors to do that. On the in vivo study, the authors may also discuss the results in the context of the literature, looking e.g. at J. Mater. Chem. B 2016 (4) 7249-7258.

In the introduction the authors should discuss further why this specific PLGA was selected and discuss electrospinning of aliphatic polyesters, e.g. with regards to J. Mech. Behav. Biomed. Mater. 2017 (65) 724-733 and ACS Appl. Bio Mater. 2019 (2) 4258–4270.

**********

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Reviewer #2: No

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Sep 29;15(9):e0239366. doi: 10.1371/journal.pone.0239366.r004

Author response to Decision Letter 1

7 Jul 2020

NOTE: The revised contents in the manuscript were labeled in RED. Our responses were written in BLUE just after each comment (BLACK) of the reviewers. The revised content of manuscript was briefly shown following our response.

First of all, we would like to express our great appreciation to the reviewers for their helpful comments and suggestions.

Reviewer #1: Dear authors, thank you for the provided responses. However it is my opinon that green staining that is seen in figure 7 depends on red blood cell unspecific fuorescence. without high magnification images I cannot say that this is acceptable.

Response: Thank you for your kind comments. The images were updated into high magification. And the positions of vascularization and microthrombus were point out with red arrows in merged images.

Reviewer #2:

1.The authors have clarified the points raised previously with additional results that support their claims. The question on the stability of the coating is still only partially answered and it would need an additional degradation test to check the kinetics of any dopamine released. I would invite the authors to do that. On the in vivo study, the authors may also discuss the results in the context of the literature, looking e.g. at J. Mater. Chem. B 2016 (4) 7249-7258.

Response: Thank you for your kind comments. The articles you recommended inspired me a lot. The release of drug is often accompanied by material degradation. However, the situation was different in the polydopamine-assisted system. The coating of polydopamine on materials was formed by the covalent bonds and strong noncovalent forces, including hydrogen bonding, charge transfer, and π-stacking. Polydopamine would usually undergo degradation in vitro in the presence of oxidizing agents such as H2O2. In vivo studies performed by Langer’s group have also demonstrated that It took 8 weeks for polydopamine implants to completely degrade. It is quite a long time to wound healing. In order to demonstrate the stability of the coating, a degradation test was conducted as described in manuscript. And the result was shown in Figure S2.

S2 Fig. Stability of coating polydopamine. The releasing test of pDA from pDA/PLGA fibers. The PLGA fibers was set as control. All data would be reported in the form of mean±standard deviation.

Page 7： ……In order to demonstrated the stability of the polydopamine coating, a test was conducted. The PLGA and pDA/PLGA fibers were respectively soaked in 3 mL PBS solution (pH 7.2). 100 μL supernatant was collected as sample at each time point and detected the absorbance at 450 nm using an auto microplate reader (Tecan M200, Switzerland) as reported.[23]……

Page 13-14：……The stability of the coating polydopamine was test. The result was shown in Figure S2. The released dopamine will form nanoparticles in aqueous solution, turning the solution to brown. The color changing can be semiquantitatively detected under OD 450. According to the result, there is no significant difference between PLGA and pDA/PLGA fibers. It indicated that dopamine barely releases from the PLGA fibers. The release of drug is often accompanied by material degradation.[24] However, the situation was different in the polydopamine-assisted system. The coating of polydopamine on materials was formed by the covalent bonds and strong noncovalent forces, including hydrogen bonding, charge transfer, and π-stacking.[25] Polydopamine would usually undergo degradation in vitro in the presence of oxidizing agents such as H2O2. In vivo studies performed by Langer’s group have also demonstrated that It took 8 weeks for polydopamine implants to completely degrade.[26] It is quite a long time to wound healing. Therefore, the absorbed PDGF-BB most likely released from fibers due to rupture of hydrogen bond between PDGF-BB and polydopamine.……

2. In the introduction the authors should discuss further why this specific PLGA was selected and discuss electrospinning of aliphatic polyesters, e.g. with regards to J. Mech. Behav. Biomed. Mater. 2017 (65) 724-733 and ACS Appl. Bio Mater. 2019 (2) 4258–4270.

Response: Thank you for your kind comments. The recent reports mentioned in the comments about the PLGA and electrospinning was quite interesting. And it has been cited in the revised manuscript to discuss why this specific PLGA was selected and discuss electrospinning of aliphatic polyesters.

Page 3:……Poly[(rac-lactide)-co-glycolide] (PLGA), a FDA-approved polyester, has been widely used in the field of regenerative medicine due to its hydrolytic degradation and good controllability of macro properties. And electrospinning is a simple technique for the efficient production of micro to nano fibers from polymer solutions with high productivity and low cost. It was believed that electrospun fibers had the potential to achieve high drug loading and the ability to sustain drug release. Shih-Feng Chou studied the loading and release of tenofovir (TFV), a hydrophilic small molecule drug, into fibers of polycaprolactone (PCL) and poly (D,L-lactic-co-glycolic) acid (PLGA). The result showed that Sustained release of TFV depended on PLGA content. The PLGA fibers has outstanding sustained-release ability.[8] Amy Contreras’ group encapsulated a photosensitizer (PS) in PCL/PLGA fibers in the PS inert state, so that the antibacterial function would be activated on-demand via a visible light source.[9] This study successfully demonstrates the significant potential of PS-encapsulated electrospun fibers as photodynamically active biomaterial for antibiotic-free infection control. These studies fully demonstrated the advantages of PLGA electrospinning in drug sustained release.……

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PLoS One. doi: 10.1371/journal.pone.0239366.r005

Decision Letter 2

Andrea Caporali

12 Aug 2020

PONE-D-19-33330R2

Polydopamine-assisted PDGF-BB immobilization on PLGA fibrous substrate enhances wound healing via regulating anti-inflammatory and cytokine secretion

PLOS ONE

Dear Dr. Zhang,

The reviewer 1 and I agreed that the CD31 staining in Figure 7 doesn't mark the endothelium; however the green fluorescent staining is due to the auto-fluorescence of erytrocytes in the vessels. We are asking to provide a better staining of the vessels. CD31 antibody can be used together with alpha-SMA to define the vessels. Alternatively, vWF or Isolecti-B4 can be used to better identify the endothelial cells.

Please submit your revised manuscript by Sep 26 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Andrea Caporali, PhD

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

**********

6. Review Comments to the Author

Reviewer #1: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Sep 29;15(9):e0239366. doi: 10.1371/journal.pone.0239366.r006

Author response to Decision Letter 2

6 Sep 2020

Reviewer：The reviewer 1 and I agreed that the CD31 staining in Figure 7 doesn't mark the endothelium; however the green fluorescent staining is due to the auto-fluorescence of erytrocytes in the vessels. We are asking to provide a better staining of the vessels. CD31 antibody can be used together with alpha-SMA to define the vessels. Alternatively, vWF or Isolecti-B4 can be used to better identify the endothelial cells.

Response: Thank you for your kind comments. The immunofluorescence images were replaced with immunohistochemical staining of CD31 with high magification. And the previous immunofluorescence images were settled in supporting information. positions of vascularization were point out with red arrows in merged images.

Page 15-16: Particularly, according to the result of CD31 staining (Fig 8), a lot of brown positive staining was observed in group of pDA/PLGA/PDGF-BB, which was believed to be neovascularization. PDA/PLGA/PDGF-BB substrate treated group showed a highly angiogenesis structure, which might be attributed to the facilitated penetration of PDGF-BB into deep dermal tissues and the prolonged residence of PDGF-BB conjugate. Most of the self-regeneration ability of skin was from basal layer [28, 29], hair follicle [29, 30] and dermis [31]. Therefore, the main task of repairing the full skin injury is to reconstruct the skin structure. It is recognized that extracellular matrix (ECM) deposition and vascularization are the most important in tissue regeneration including skin. PDGF has been accepted to stimulate the proliferation of various cells and the production of ECM [32]. In addition, the PDGF stimulates human ﬁbroblasts to contract ﬂoating collagen matrices [33]. In the process of tissue remodeling, PDGF helps to adjust the mitotic activity or the productivity of collagen in dermal ﬁbroblasts [34]. To vascularization, it is worth noting that the effect of PDGF is weaker than that of FGF and VEGF and is not essential for the initial formation of blood vessels [15]. But PDGF is particularly important in blood vessel maturation. The presence of the neovascularization indicated that the vessels were much more mature to take on more blood supply in group of pDA/PLGA/PDGF-BB. In vivo experiments have shown that PDGF is critical for the recruitment of pericytes, thereby increasing the structural integrity of blood vessels [32]. In this study, it was found that the immobilized PDGF-BB greatly enhanced ECM maturation and vascularization, and provide the necessary ECM and vascular structure for skin regeneration.

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PLoS One. doi: 10.1371/journal.pone.0239366.r007

Decision Letter 3

Andrea Caporali

7 Sep 2020

Polydopamine-assisted PDGF-BB immobilization on PLGA fibrous substrate enhances wound healing via regulating anti-inflammatory and cytokine secretion

PONE-D-19-33330R3

Dear Dr. Zhang,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Andrea Caporali, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0239366.r008

Acceptance letter

Andrea Caporali

17 Sep 2020

PONE-D-19-33330R3

Polydopamine-assisted PDGF-BB immobilization on PLGA fibrous substrate enhances wound healing via regulating anti-inflammatory and cytokine secretion

Dear Dr. Zhang:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Appearance of materials.

The appearance photographs of (A) PLGA and (B)pDA/PLGA fibers.

(TIF)

Click here for additional data file.^{(3.5MB, tif)}

S2 Fig. Stability of coating polydopamine.

The releasing test of pDA from pDA/PLGA fibers. The PLGA fibers was set as control. All data would be reported in the form of mean±standard deviation.

(TIF)

Click here for additional data file.^{(4.4MB, tif)}

S3 Fig. Immunofluorescence staining of CD31.

Effects of the substitutes on neovascularization of wound area at day 7 after transplantation by examining the expression of CD31. (The bars = 50 μm).

(TIF)

Click here for additional data file.^{(7.4MB, tif)}

S1 Table. Statistic of fiber diameter of each sample.

Statistic of fiber diameter of each sample. All data would be reported in the form of mean±standard deviation.

(DOCX)

Click here for additional data file.^{(14.9KB, docx)}

S1 File

(RAR)

Click here for additional data file.^{(267.7KB, rar)}

Attachment

Submitted filename: Responses to Reviewers.doc

Click here for additional data file.^{(51.1MB, doc)}

Attachment

Submitted filename: Responses to Reviewers.doc

Click here for additional data file.^{(10MB, doc)}

Attachment

Submitted filename: Responses to Reviewers.doc

Click here for additional data file.^{(36.1MB, doc)}

Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

[pone.0239366.ref001] 1.Bottcher-Haberzeth S, Biedermann T, Reichmann E. Tissue engineering of skin. Burns. 2010;36(4):450–60. 10.1016/j.burns.2009.08.016 PubMed PMID: WOS:000277856300002. [DOI] [PubMed] [Google Scholar]

[pone.0239366.ref002] 2.Supp DM, Boyce ST. Engineered skin substitutes: practices and potentials. Clin Dermatol. 2005;23(4):403–12. 10.1016/j.clindermatol.2004.07.023 PubMed PMID: WOS:000231045600013. [DOI] [PubMed] [Google Scholar]

[pone.0239366.ref003] 3.Mansbridge JN. Tissue-engineered skin substitutes in regenerative medicine. Curr Opin Biotech. 2009;20(5):563–7. 10.1016/j.copbio.2009.08.008 PubMed PMID: WOS:000272712000009. [DOI] [PubMed] [Google Scholar]

[pone.0239366.ref004] 4.MacNeil S. Biomaterials for tissue engineering of skin. Mater Today. 2008;11(5):26–35. 10.1016/S1369-7021(08)70087-7 PubMed PMID: WOS:000255691600020. [DOI] [Google Scholar]

[pone.0239366.ref005] 5.Dixit S, Baganizi DR, Sahu R, Dosunmu E, Chaudhari A, Vig K, et al. Immunological challenges associated with artificial skin grafts: available solutions and stem cells in future design of synthetic skin. J Biol Eng. 2017;11. doi: UNSP 49 10.1186/s13036-017-0089-9 PubMed PMID: WOS:000417878800001. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0239366.ref006] 6.Kamel RA, Ong JF, Eriksson E, Junker JPE, Caterson EJ. Tissue Engineering of Skin. J Am Coll Surgeons. 2013;217(3):533–55. 10.1016/j.jamcollsurg.2013.03.027 PubMed PMID: WOS:000324043300019. [DOI] [PubMed] [Google Scholar]

[pone.0239366.ref007] 7.Wood FM. Skin regeneration: The complexities of translation into clinical practise. Int J Biochem Cell B. 2014;56:133–40. 10.1016/j.biocel.2014.10.025 PubMed PMID: WOS:000347127400016. [DOI] [PubMed] [Google Scholar]

[pone.0239366.ref008] 8.Chou SF, Woodrow KA. Relationships between mechanical properties and drug release from electrospun fibers of PCL and PLGA blends. J Mech Behav Biomed. 2017;65:724–33. 10.1016/j.jmbbm.2016.09.004 PubMed PMID: WOS:000390625100067. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0239366.ref009] 9.Amy Contreras MJR, Wood Simon, Jessica D. Schiffman, Tronci Giuseppe. Photodynamically Active Electrospun Fibers for Antibiotic-Free Infection Control. ACS Appl Bio Mater. 2019;2:4258–70. 10.1021/acsabm.9b00543 [DOI] [PubMed] [Google Scholar]

[pone.0239366.ref010] 10.Schneider RK, Anraths J, Kramann R, Bornemann J, Bovi M, Knuchel R, et al. The role of biomaterials in the direction of mesenchymal stem cell properties and extracellular matrix remodelling in dermal tissue engineering. Biomaterials. 2010;31(31):7948–59. 10.1016/j.biomaterials.2010.07.003 PubMed PMID: WOS:000282109100009. [DOI] [PubMed] [Google Scholar]

[pone.0239366.ref011] 11.Zhou T, Sui BY, Mo XM, Sun J. Multifunctional and biomimetic fish collagen/bioactive glass nanofibers: fabrication, antibacterial activity and inducing skin regeneration in vitro and in vivo. Int J Nanomed. 2017;12:3495–507. 10.2147/Ijn.S132459 PubMed PMID: WOS:000401001900002. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0239366.ref012] 12.Song JF, Zhang PF, Cheng L, Liao Y, Xu B, Bao R, et al. Nano-silver in situ hybridized collagen scaffolds for regeneration of infected full-thickness burn skin. J Mater Chem B. 2015;3(20):4231–41. PubMed PMID: WOS:000354450300013. 10.1039/c5tb00205b [DOI] [PubMed] [Google Scholar]

[pone.0239366.ref013] 13.Jin GR, Prabhakaran MP, Kai D, Ramakrishna S. Controlled release of multiple epidermal induction factors through core-shell nanofibers for skin regeneration. Eur J Pharm Biopharm. 2013;85(3):689–98. 10.1016/j.ejpb.2013.06.002 PubMed PMID: WOS:000330200400043. [DOI] [PubMed] [Google Scholar]

[pone.0239366.ref014] 14.Fleischli JG, Laughlin TJ. Electrical stimulation in wound healing. The Journal of foot and ankle surgery: official publication of the American College of Foot and Ankle Surgeons. 1997;36(6):457–61. 10.1016/s1067-2516(97)80099-3 . [DOI] [PubMed] [Google Scholar]

[pone.0239366.ref015] 15.Barrientos S, Stojadinovic O, Golinko MS, Brem H, Tomic-Canic M. Growth factors and cytokines in wound healing. Wound repair and regeneration: official publication of the Wound Healing Society [and] the European Tissue Repair Society. 2008;16(5):585–601. 10.1111/j.1524-475X.2008.00410.x . [DOI] [PubMed] [Google Scholar]

[pone.0239366.ref016] 16.Allen RJ Jr., Soares MA, Haberman ID, Szpalski C, Schachar J, Lin CD, et al. Combination therapy accelerates diabetic wound closure. PloS one. 2014;9(3):e92667 10.1371/journal.pone.0092667 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0239366.ref017] 17.Xie Z, Paras CB, Weng H, Punnakitikashem P, Su LC, Vu K, et al. Dual growth factor releasing multi-functional nanofibers for wound healing. Acta Biomater. 2013;9(12):9351–9. 10.1016/j.actbio.2013.07.030 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0239366.ref018] 18.Park JW, Hwang SR, Yoon IS. Advanced Growth Factor Delivery Systems in Wound Management and Skin Regeneration. Molecules. 2017;22(8). 10.3390/molecules22081259 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0239366.ref019] 19.Wang Y, Fu C, Wu Z, Chen L, Chen X, Wei Y, et al. A chitin film containing basic fibroblast growth factor with a chitin-binding domain as wound dressings. Carbohydrate polymers. 2017;174:723–30. 10.1016/j.carbpol.2017.05.087 . [DOI] [PubMed] [Google Scholar]

[pone.0239366.ref020] 20.Lee JS, Lee JC, Heo JS. Polydopamine-assisted BMP-2 immobilization on titanium surface enhances the osteogenic potential of periodontal ligament stem cells via integrin-mediated cell-matrix adhesion. Journal of cell communication and signaling. 2018;12(4):661–72. 10.1007/s12079-018-0468-0 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0239366.ref021] 21.Davoudi P, Assadpour S, Derakhshan MA, Ai J, Solouk A, Ghanbari H. Biomimetic modification of polyurethane-based nanofibrous vascular grafts: A promising approach towards stable endothelial lining. Materials science & engineering C, Materials for biological applications. 2017;80:213–21. 10.1016/j.msec.2017.05.140 . [DOI] [PubMed] [Google Scholar]

[pone.0239366.ref022] 22.Wang Z, Chen L, Wang Y, Chen X, Zhang P. Improved Cell Adhesion and Osteogenesis of op-HA/PLGA Composite by Poly(dopamine)-Assisted Immobilization of Collagen Mimetic Peptide and Osteogenic Growth Peptide. ACS applied materials & interfaces. 2016;8(40):26559–69. 10.1021/acsami.6b08733 . [DOI] [PubMed] [Google Scholar]

[pone.0239366.ref023] 23.Zhang C, Miyatake H, Wang Y, Inaba T, Wang Y, Zhang P, et al. A Bioorthogonal Approach for the Preparation of a Titanium-Binding Insulin-like Growth-Factor-1 Derivative by Using Tyrosinase. Angewandte Chemie. 2016;55(38):11447–51. 10.1002/anie.201603155 . [DOI] [PubMed] [Google Scholar]

[pone.0239366.ref024] 24.Tronci G, Yin J, Holmes RA, Liang H, Russell SJ, Wood DJ. Protease-sensitive atelocollagen hydrogels promote healing in a diabetic wound model. J Mater Chem B. 2016;4(45):7249–58. 10.1039/c6tb02268e PubMed PMID: WOS:000388894600007. [DOI] [PubMed] [Google Scholar]

[pone.0239366.ref025] 25.Liu YL, Ai KL, Lu LH. Polydopamine and Its Derivative Materials: Synthesis and Promising Applications in Energy, Environmental, and Biomedical Fields. Chem Rev. 2014;114(9):5057–115. 10.1021/cr400407a PubMed PMID: WOS:000336078200013. [DOI] [PubMed] [Google Scholar]

[pone.0239366.ref026] 26.Bettinger CJ, Bruggeman PP, Misra A, Borenstein JT, Langer R. Biocompatibility of biodegradable semiconducting melanin films for nerve tissue engineering. Biomaterials. 2009;30(17):3050–7. 10.1016/j.biomaterials.2009.02.018 PubMed PMID: WOS:000266194900003. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0239366.ref027] 27.Lederle W, Stark HJ, Skobe M, Fusenig NE, Mueller MM. Platelet-derived growth factor-BB controls epithelial tumor phenotype by differential growth factor regulation in stromal cells. The American journal of pathology. 2006;169(5):1767–83. 10.2353/ajpath.2006.060120 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0239366.ref028] 28.Fuchs E. Skin stem cells: rising to the surface. The Journal of cell biology. 2008;180(2):273–84. 10.1083/jcb.200708185 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0239366.ref029] 29.Blanpain C, Fuchs E. Epidermal stem cells of the skin. Annu Rev Cell Dev Bi. 2006;22:339–73. 10.1146/annurev.cellbio.22.010305.104357 PubMed PMID: WOS:000242325100015. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0239366.ref030] 30.Brockmann I, Ehrenpfordt J, Sturmheit T, Brandenburger M, Kruse C, Zille M, et al. Skin-Derived Stem Cells for Wound Treatment Using Cultured Epidermal Autografts: Clinical Applications and Challenges. Stem Cells Int. 2018. doi: Artn 4623615 10.1155/2018/4623615. PubMed PMID: WOS:000429623300001. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0239366.ref031] 31.Lorenz K, Sicker M, Schmelzer E, Rupf T, Salvetter J, Schulz-Siegmund M, et al. Multilineage differentiation potential of human dermal skin-derived fibroblasts. Exp Dermatol. 2008;17(11):925–32. 10.1111/j.1600-0625.2008.00724.x PubMed PMID: WOS:000259936100004. [DOI] [PubMed] [Google Scholar]

[pone.0239366.ref032] 32.Lin H, Chen B, Sun W, Zhao W, Zhao Y, Dai J. The effect of collagen-targeting platelet-derived growth factor on cellularization and vascularization of collagen scaffolds. Biomaterials. 2006;27(33):5708–14. 10.1016/j.biomaterials.2006.07.023 . [DOI] [PubMed] [Google Scholar]

[pone.0239366.ref033] 33.Rhee S, Grinnell F. P21-activated kinase 1: convergence point in PDGF- and LPA-stimulated collagen matrix contraction by human fibroblasts. The Journal of cell biology. 2006;172(3):423–32. 10.1083/jcb.200505175 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0239366.ref034] 34.Jinnin M, Ihn H, Mimura Y, Asano Y, Yamane K, Tamaki K. Regulation of fibrogenic/fibrolytic genes by platelet-derived growth factor C, a novel growth factor, in human dermal fibroblasts. Journal of cellular physiology. 2005;202(2):510–7. 10.1002/jcp.20154 . [DOI] [PubMed] [Google Scholar]

PERMALINK

Polydopamine-assisted PDGF-BB immobilization on PLGA fibrous substrate enhances wound healing via regulating anti-inflammatory and cytokine secretion

Xiao Yang

Peng Zhan

Xiuyan Wang

Qiushuang Zhang

Yi Zhang

Haitao Fan

Ranwei Li

Ming Zhang

Roles

Abstract

Introduction

Materials and methods

Materials

Electrospinning of fibers

PDA-assisted immobilization of PDGF-BB onto fibers

Surface characterization

Animals and experimental protocol

Immunohistochemistry and immunofluorescence

Cytokine analysis

Statistical analysis

Results and discussions

Surface characterization of modified fibers

Fig 1. Scanning electron microscope (SEM) photo.

Fig 2. FI-TR spectrum.

Fig 3. XPS analyses.

Table 1. Surface chemical composition of XPS.

Fig 4. The hydrophilia performance of the PLGA fiber substrates.

Kinetics of absorption and release

Fig 5. Kinetics of growth factor absorption and release.

In vivo assessment of pDA/PLGA/PDGF-BB substrate

Fig 6. Macroscopic appearance of wounds.

Fig 7. H&E staining of the tissue.

Fig 8. Immunohistochemical staining of CD31.

Cytokine analysis

Fig 9. Cytokine analysis by ELISA method.

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Andrea Caporali

Roles

Author response to Decision Letter 0

Decision Letter 1

Andrea Caporali

Roles

Author response to Decision Letter 1

Decision Letter 2

Andrea Caporali

Roles

Author response to Decision Letter 2

Decision Letter 3

Andrea Caporali

Roles

Acceptance letter

Andrea Caporali

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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