Space-Oriented Nanofibrous Scaffold with Silicon-Doped Amorphous

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A space-oriented nanofibrous scaffold with Silicon-doped amorphous calcium phosphate nanocoating for diabetic wound healing Yuqi Jiang, Yiming Han, Jie Wang, Fang Lv, Zhengfang Yi, Qinfei Ke, and He Xu ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00657 • Publication Date (Web): 10 Jan 2019 Downloaded from http://pubs.acs.org on January 20, 2019

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ACS Applied Bio Materials

A space-oriented nanofibrous scaffold with Silicon-doped amorphous calcium phosphate nanocoating for diabetic wound healing Yuqi Jiang a,1, Yiming Hanb,1, Jie Wanga, Fang Lvb, Zhengfang Yib,*, Qinfei Ke a,*,

He Xua,*

a College

of Chemistry and Materials Sciences, Shanghai Normal University,

No. 100 Guilin Road, Shanghai 200234, China b Shanghai

Key Laboratory of Regulatory Biology, Institute of Biomedical

Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China *Corresponding authors. 1

The two authors contributed to the work equally.

E-mail addresses: [email protected] (H. Xu), [email protected] (Q. Ke), [email protected] (Z. Yi)

ABSTRACT: Insufficient angiogenesis always leads to the prolonged or even non-healing process for diabetic wound. Hence, the stimulation of angiogenesis in the wound site is an effective therapeutic strategy for the treatment of diabetic wound. In this study, we reported a spaced-oriented

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electrospun scaffold with Silicon-doped amorphous calcium phosphate nanocoating on the surface (Si-ACP/PM). The results indicated that the Si-ACP/PM

scaffold

exhibited

an

alternately

random

and

aligned

microstructure, most of the nanofibers densely deposited in a random manner to from columnar embossments, while others loosely aligned between the embossments to construct a three-dimensional porous microstructure. Furthermore, the Si-ACP nanoparticles with the diameter of about 40 nm were well distributed on the surface of each nanofiber and the Si ions could sustain release from the scaffold. Results revealed that the Si-ACP/PM scaffold can promote the proliferation and migration of human umbilical vein endothelial cells (HUVECs) in vitro. The in vivo study further demonstrated that the Si-ACP scaffold effectively accelerated the wound healing via the promotion of angiogenesis, collagen deposition as well as re-epithelialization in the diabetic wound bed. Taken together, the study indicates that the spaced-oriented electrospun scaffold with Silicon-doped amorphous calcium phosphate nanocoating on the surface, which could significantly stimulate the angiogenesis during the process of diabetic wound healing shows a great potential for the therapy of diabetic wound.

KEYWORDS: electrospinning, micropattern, nanocoating, angiogenesis, diabetic wound healing 1.

Introduction


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Diabetes mellitus (DM), a metabolic diseases with an increasing number of patients, seriously affects the quality of patients ’ life.1 Diabetic foot ulcer (DFU) is one of the most severe complications of DM.2 Due to the long-term hyperglycemia stimulation, it is difficult for the newly vessel formation, which will lead to the deficiency in the transportation of oxygen and nutrients to the wound site and ultimately delay the process of wound healing.3-4 Conventional treatments of diabetic wound include surgical debridement and wound dressing nursing. However, most of the wound dressings used in clinical usually lack of bioactivity, which could not effectively promote the healing of diabetic wound.5 Therefore, it is expected to design an innovative skin tissue engineering scaffold with high bioactivity that can accelerate the diabetic wound healing via the stimulation of angiogenesis in the wound site but also accelerate the diabetic wound healing, it would have a good prospect for application in skin tissue engineering. The nanofibrous scaffolds obtained via electrospinning possess large specific surface area, highly interconnected porous structure and controllable surface morphologies. Beyond that, the nanofibrous structure of electrospun fiber is similar to the native structure of extracellular matrix (ECM) which could create a biomimetic microenvironment for cells adhesion and proliferation. In recent years, electrospun scaffold has been drawn increasing attention in the field of skin wound healing.6-7 The traditional electrospun nanofibrous scaffold which is collected by a flat collector exhibit a nonwoven structure. As the


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surface topography of scaffolds has a significant effect on cell behaviors such as adhesion, proliferation and even differentiation,8-10 in our previous work, through controlling the surface morphology of collector during the process of electrospinning, we have successfully designed and prepared a variety of micro-patterned

nanofibrous

scaffolds.11-12

Compared

with

randomly

deposited scaffold, the micro-patterned scaffold exhibits higher porosity, which is beneficial for nutrients and oxygen transfer and could promote cell-cell connection and cell ingrowth. Among them, the spaced-oriented electrospun scaffold, which has been reported in our recent study, could significantly

stimulate

the

proliferation

and

migration

of

HUVECs.

Furthermore, cell cultured on such scaffold exhibited a great angiogenic ability with the cell cytoskeleton and nuclei remodeling.12-14 Hence, it’s reasonable to hypothesis that the electrospun scaffold with this kind of spaced-oriented microstructure might be suitable for the treatment of DFU. In the other hand, as most of the micro-patterned electrospun scaffolds are prepared by synthetic polymer, which are always lack of bioactivity and the hydrophobic surface remain insufficiently effective to support cell growth. Although various bioactive substance such as cytokines, protein and bioactive molecules have been incorporated into scaffolds to enhance their bioactivity,15-17 these bioactive compounds display distinct shortcomings, including high cost, burst release and instability. Recently, the strategy of utilizing inorganic bioactive agents to improve the bioactivity of scaffolds has


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attracted increasing interest.18-20 Amorphous calcium phosphate (ACP), a kind of calcium orthophosphate salts, has been wildly applied in biomedical fields due to its excellent bioactivity.21-23 The amorphous structure of ACP make the Calcium ions can be easily sustained release from ACP after implanted. It has been demonstrated that Ca ions play important roles in inflammatory cell infiltration, fibroblast proliferation and keratinocyte migration,24-25 which are closely related to the process of wound healing.26-28 Therefore, the ACP may have a potential application for the diabetic wound healing. On the other hand, it has been proved that Si4+ could mediate cell behavior and significantly promote angiogenic differentiation of endothelial cells.20, 29 Hence, silicon can be doped into ACP to further improve the angiogenesis-stimulation capacity of ACP. If the Si doped ACP (Si-ACP) is incorporated into the spaced-oriented electrospun scaffolds, this new kind of scaffolds will greatly improve the therapeutic efficiency of diabetic wound. Pulsed laser deposition (PLD) is a versatile technology to prepare uniform nanosized inorganic film and the stoichiometry as well as crystallinity of the deposited film can be finely controlled though regulating the process parameter.30-31 By using this method, the surfaces of polymeric scaffolds can be conveniently modified with uniformly nanosized coatings at room temperature. Researches have demonstrated that after shining a laser onto HA at room temperature, HA will uniformly deposit on to the substrate materials in the form of amorphous calcium phosphate (ACP) coatings.32-34


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Therefore, to prepare a uniform Si-ACP coating on the surface of the spaced-oriented electrospun scaffold, the PLD surface modification strategy could be applied and the silicon doped hydroxyapatite is chosen as a target during

this

process.

Both

Poly

(D,

L-lactic

acid)

(PDLLA)

and

Polycaprolactone (PCL) are biocompatible polymer and have been widely used for preparing electrospun scaffolds. Our previously study had proved that the combination of PDLLA and PCL possessed a better pattern formation ability during the electrospinning process and mechanical stability in wet condition.13 Hence, in the present study, we designed and fabricated a composite spaced-oriented PDLLA/PCL electrospun scaffold by the modified electrospinning method, and then the highly bioactive Si-ACP nanocoating were deposited on the surface of each nanofiber by using the PLD strategy. As the Si-ACP nanocoating can not only improve the wettability of the scaffold, but also can offer beneficial chemical cues for cells, it is hypothesized that the combination of the spaced-oriented microstructure and the Si-ACP nanocoating on the surface of electrospun scaffold might synergistically accelerate the diabetic wound healing. 2.

Materials and Methods Materials. Poly (D, L-lactic acid) (PDLLA, Mw = 45 kDa) was supplied by

Jinan Daigang Biomaterial Co., Ltd. (Shandong, China). Polycaprolactone (PCL, Mw=80000) was purchased from Sigma-Aldrich (Saint Louis, MO,


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USA). N, N-Dimethylformamide (DMF), Tetrahydrofuran (THF) and Tetraethyl orthosilicate (TEOS) were purchased from Aladdin Reagent Co.. Calcium nitrate tetrahydrate (Ca(NO3)2·4H2O), Ammonium phosphate ((NH4)2·HPO4) and ammonium hydroxide (NH4OH) was obtained from Richjoint Chemical Reagent Co., Ltd. (Shanghai, China). Preparation and characterization of silica doped hydroxyapatite bulk. HAp and Silicon doped HAp (Ca10(PO4)6-x(SiO4)x(OH)2-x,) with 2.4% mass fraction of Si were synthesized via wet chemical method. Briefly, Ca (NO3)2·4H2O (0.08 mol) was dissolved in distilled water (300 mL) and TEOs (1.53 mL) was added into the solution. Then, 200 mL (NH4)2·HPO4 (0.256 mol/L) was dropwise added into the above solution under stirring in an oil bath system (90 ℃). The pH value was adjusted to 11 during the reaction. After aging for 6 h, the product was isolated by centrifugation and dried at 60 ℃ overnight. Then the dry powers were calcined at 900 ℃ for 3 h. The Si-HAp ceramic disk (Ø 25 × 5 mm) was obtained by compacting the powders under table uniaxial pressure (20 MPa) and sintering at 1250 ℃ for 3h. The morphology of the Si-HAp sample was observed by scanning electron microscopy (FE-SEM, HitachiS-4800, CanSan). The compositional analysis of the sample was performed by energy dispersive spectroscopy (EDS). X-ray diffraction was used to analyzed the crystal structure of the sample (XRD, D/max-IIB, Japan).


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Preparation and characterization of Si-ACP/PM. The nanofibrous membrane with spaced-oriented structure (PM) was fabricated by the electrospinning technique. In a typical experimental procedure, the electrospun solution was prepared by dissolving the blend of PDLLA and PCL (m/m=4/1) in the mixed solution of DMF and THF (V/V=4/1) at a concentration of 6%. The solution was electrospun by a syringe pump (LSP01-1A, Baoding longer Precision Pump, China) with the flow rate of 0.02 mL/min. The voltage applied to the needle was 8 kV. The distance between the tip of needle and the collector was 12 cm. During the process of electrospinning, a special wire spring was applied as a collector. The parameters of the wire spring are as follows: the interval distance is 1mm, the external diameter and the wire diameter are 2 cm and 0.3 mm, respectively. After electrospinning, the resulting electrospun scaffolds were kept at 37° in a vacuum oven for 1 day. The nanocoating on the surface of PM was fabricated by using the PLD method. The as-prepared Si-HAp bulk were used as the target. The prepared PM was fixed in vacuum chamber of the PLD instrument. The Si-HAp discs were set as a target and irradiated by baser beam. The parameters of the PLD process were set as follows: the repetition rate was 5 Hz, the laser fluency was 160 MJ, the target-substrate distance was 50 mm, the O2 ambient pressure was 20 MPa and the deposition time was 40 min. The obtained composite membranes were named as Si-ACP/PM.


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The surface morphology of the composite scaffold was analyzed via field-emission scanning electron microscopy (FE-SEM, HitachiS-4800, CanScan) and optical microscope. The surface wettability of composite scaffolds was detected by a water contact angle method (Kruss GmbH DSA 100 Mk 2). The in vitro silicon ions release profile was conducted according to our previous work.35, 38 The concentration of silicon ions in the released medium was detected by inductively coupled plasma atomic emission spectrometry (ICP-AES, Perkin-Elmer Optima 7000 DV). Cell culture. The Human Umbilical Vein Endothelial Cells (HUVECs) and Human Aortic Fibroblasts (HAFs) were obtained from the cell bank of East China Normal University and incubated in Dulbecco’s Modified Eagle’s Medium (DMEM, HyClone, Logan, Utah, USA), which supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were cultured in a humidified incubator supplied with 5% CO2 at 37℃. Cell proliferation assay. The viability of cells cultured on the composite scaffolds was investigated by cell counting kit assay (CCK-8) at 1, 3 and 7 days, respectively. HUVECs and HSFs (1 × 104 cells per well) were seeded on the samples in 48-well plates with exposure to5% CO2 at 37℃. Then, at each time point, after the medium was removed, the CCK-8 contained DMEM was added and incubated at 37℃ for 1h. The absorbance at 450 nm by microplate reader at 450 nm. (Epoch, BIO-TEK, USA)


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Cells migration and tube formation assay. The effect of the composite scaffolds on HUVECs migration was evaluated with a Transwell assay according to our previous work.35 Briefly, 5×104 HUVECs were cultured on the upper chamber of 24-well plate and supplied with 100 μl serum-free medium. The composite scaffold was placed in the bottom of the chamber and incubated with ECM medium. After culturing for 12 h, 4% paraformaldehyde was used to fixed the invaded cells, and then stained with crystal violet (0.1% w/v). the invaded cells were observed by Microscope (Olympus Optical Co, Ltd, Tokyo, Japan). Tube formation assay was conducted according to our previous work.35 Matrigel (BD Biosciences) was coated on each well of the pro-cooling 24-well at 100 μl/well. Then, HUVECs were seeded with the density of 2×104 per well and co-cultured with different scaffolds. After 6 h of co-incubation in a humidified incubator at 37 °C, 5% CO2, the tube formation abilities of endothelial cells were evaluated by counting the number of nodes and tube length in the optical images obtained through a microscope. Wound healing assessment. A diabetic wound model was established to assess the effect of the composite scaffolds on the diabetic wound. The experimental was performed in the Specific Pathogen Free (SPF) environment and all the experimental procedures involving mice were given approval by the Animal Investigation Committee of the Institute of Biomedical Sciences and School of Life Sciences, East China Normal University.


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Diabetic was induced by intraperitoneally injecting streptozotocin according to our previously decscription.32 In this study, the diabetic mice (12-14week) were randomly divided into four groups (Control, PM, ACP/PM and Si-ACP/PM) and the dorsal hair of mice was shaved under inhalation isoflurane anesthesia (5%). A round full thickness shin wound (about 8 mm diameter) was created on the dorsum of each rat. Three kind of to-be-tested scaffold (PM, ACP/PM and Si-ACP/PM) with the same size were covered on the wound. The blank control group was left untreated (CTR). The wound areas in all mice were photographed on day 0, 5, 7, 9, 13 and calculated by Image J. RT-PCR analysis of the wound tissue. The effect of the composite scaffold on the expression of angiogenesis related genes was detected by RT-PCR according to our previous report.35 Briefly, the total RNA of the regenerated tissue was extracted by Trizol according to manufacturer’s instructions. cDNA was synthesized by reverse transcribe using Prime Script

TM

RT Master Mix

(Takara Bio Inc., Shiga, Japan). SYBR Green detection reagent (Takara Bio Inc., Shiga, Japan) was used to conduct Q-PCR. The primer sequences were shown in Table 1. The gene expression level was normalized to β-Actin.

Table 1. Primer Sequences Used in Q-RT-PCR Gene

Primer


11


Actin

5’-GTACGCCAACACAGTGCTG-3’

TGF-β

5’-TCCCAACTACAGGACCTTTTTCA-3’

VEGF

5’-GAGCCTTGCCTTGCTGCTCTAC-3’

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Histological analysis. The tissues were fixed with paraformaldehyde and then embedded into paraffin block to prepare tissue sections. The effect of composite scaffold on collagen formation was analysis by Masson’s trichrome staining. The tissue sections were deparaffinized and rehydrated in a graded series of ethanol (70-100%), and then stained with Masson Trichrome Staining kit. The CD31 immunofluorescence staining was perform to assess the vascular formation in the diabetic wound beds. The sections were deparaffinized and boiled in sodium citrate buffer. For immunofluorescence staining, the sections were incubated with the primary antibodies (Abcam) at 4 °C overnight, and then the secondary antibodies were added and incubated for 2 h at room temperature. The cell nuclei were counterstained with DAPI solution (5 mg/mL). Finally, the pictures were obtained by using a fluorescence microscope (Leica Confocal microscope) and analyzed by Image J. Statistical analysis. All results were obtained from at least three independent experiments. The statistical differences were determined by one-way ANOVA firstly and then the difference between each two groups was evaluated by


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standard two-tailed Student’s t-test. The significance was considered at p < 0.05 (*), p < 0.01 (**) or p < 0.001 (***). 3. Result Characterization of silica doped hydroxyapatite and Si-ACP/PM. As shown in Fig. S1, the Si-HAp nanoparticles with defined silicon concentrations had been successfully fabricated. The SEM images in Fig. S1A showed that the HAp and Si-HAp particles presented a rod-like structure with 100 nm in length and 20 nm in width. The characteristic peaks of Si in the Energy dispersive spectrometry analysis (EDS) spectrum of Si-HAp samples confirmed the incorporation of Si inside the framework, while only O, P and Ca elements were detected in the HAp samples (Fig. S1B). As shown in Fig. S2, both HAp and Si doped HAp exhibited quite similar XRD patterns, which is corresponding to the hydroxyapatite phase (standard card of hydroxyapatite, JCPDS 09-0432). With the silicon doped, the intensity of diffraction peaks decreased, suggesting the existence of impurity phases in the samples. Fig. 1 showed the patterned composite scaffolds prepared by the modified electrospinning method and PLD technology. As observed from the optical microscopic image (Fig. 1A1-C1), the scaffolds exhibited well-organized topological morphologies with spaced-oriented structures, a lot of fibers in the scaffolds deposited randomly to form columnar embossments, while there was a part of fibers suspended between the embossments. The


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corresponding magnified scanning electron microscopy (SEM) images further showed that the nanofibers between the embossments revealed quite aligned structures (Fig. 1A2-C2). Fig. 1A2 and A3 showed a quite smooth surface of nanofiber in the PM scaffold. However, after the surface treatment by the PLD technology, a well-distributed layer with uniformed ACP nanoparticles could be clearly observed on the surface of nanofibers both in ACP/PM (Fig. 1B2 and B3) and Si-ACP/PM (Fig. 1C2 and C3) scaffolds. Besides, the addition of nanocoating layers on the fibers had no influence on the micropatterned morphologies of composite scaffolds (Fig. 1B1 and C1). The surface wettability of the composite scaffolds was investigated by water contact angle measurements. Fig. 1A4-C4 showed the water contact angles of scaffolds reduced from 127° to about 20° after being modified by ACP nanocoating, indicating that the hydrophily of the PM had been largely improved.


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Figure 1. Optical images (A1-C1), SEM images (A2-C2), the high-magnification SEM images (A3-C3) and water contact angles (A4-C4) of the composite scaffold: patterned membrane (PM) (A1-A4), PM coated with ACP (B1-B3) and PM coated with Si-ACP (C1-C3). The cumulative release of Si ions from Si-ACP/PM. The ICP analysis in Fig. 2 revealed the cumulative concentrations of Si ions released from Si-ACP/PM scaffold. The Si ions concentration released from the Si-ACP/PM group in the first day was 0.523 μg/ mL. With the increment of time, the concentration of Si ions released from the scaffold was increased. At 7 days, the cumulative release of Si ions was up to 2.13 μg/ mL.

Figure. 2 Si ions release profiles of Si-ACP/PM The effect of the Si-ACP/PM on HUVECs proliferation, migration and tube forming in vitro. The proliferation behavior of HUVECs on PM, ACP/PM and Si-ACP/PM scaffolds were shown in Fig. 3C. It was shown that the number of cells was increased with the cultural time for all the groups. There was no


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significant difference among the proliferations of cells on the three kinds of scaffolds at day 1. However, at day 3 and day 7, the number of cells cultured on the Si-ACP/PM scaffold was significantly higher than that of the other two kinds of scaffolds. The transwell assay was used to assess the HUVECs migration when co-cultured with different scaffolds (Fig. 3A and D). The results were shown that the number of invasion cells in the group cultured with Si-ACP/PM scaffolds was significantly higher than the other groups. The effect of the composite scaffold on the pro-angiogenic of HUVECs was evaluated by tubular formation assay. The HUVECs trend to formed the networks with the structures of capillary on Matrigel after 6 h in all groups (Fig. 3D). Although there was no sharp difference among the three groups in the quantitative analysis of tube length (Fig. 3E), the group of Si-ACP/PM showed a significant higher in the number of nodes than the other groups (Fig. 3E).


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Figure 3. (A) The transwell assay of HUVECs co-cultured with different scaffolds after 12 hours and (D) the corresponding quantitative analysis of migrated cells. (B) The tube formation assay of HUVECs co-cultured with different scaffolds after 6 hours of culture. (C) Proliferation of HUVECs after 1, 3 and 7 days of culture with different scaffolds. Quantitation of the tube length (E) and number of nodes (F) formed after 6 h of culture in the tube formation assay, respectively. (*P < 0.05, **P< 0.01, ***P< 0.001) The Si-ACP/PM accelerating the process of diabetic wound healing. To evaluate the effect of the composite scaffolds on the diabetic wound in vivo, a full thickness wound was created on the back of diabetic mice and treated


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with PM, ACP/PM and Si-ACP/PM membranes and blank group with undressed was set as control. It was showed that the wound area in all four groups achieved obvious reduction with increasing time (Fig. 4A and B). Although there was no significant difference among the four groups during the early stages of healing (within 5 days), the Si-ACP/PM showed a markedly higher wound healing rate than the control, PM and ACP/PM groups after 7 days. Differing from the control group (61.7%), the wound sizes of the groups treated with PM, ACP/PM and Si-ACP/PM scaffolds were reduced to 70.5%, 71.2% and 75.6% at 9 days, respectively. At day 15, the wound cover ratios in ACP/PM and Si-ACP/PM groups was 92.1% and 96.3%, respectively, which were remarkably greater than the control (76.3%) and PM groups (84.1%) (Fig. 4C). In particularly, the Si-ACP/PM scaffold showed the highest potential for accelerated diabetic wound healing among the three kinds of scaffolds


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Figure 4. (A) Representative images of the wound with different treatments group (control, PM, ACP/PM and Si-ACP/PM) at 0, 5, 7, 9 and 13 days post-surgery. (B) The trace of wound region from day 0 (blue) to days n (yellow). (C) Statistical analysis of the area of wound area in each group at day 0, 5, 7, 9 and 13 days post-surgery. (*P < 0.05, **P< 0.01, ***P< 0.001) The

Si-ACP/PM

promotes

angiogenesis

in

diabetic

wounds.

The

immunofluorescence staining of CD31 and RT-PCR analysis of the angiogenesis-related genes expression were conducted to evaluate the angiogenesis in the wound beds of the four groups. As shown in Fig. 5A and B, the CD31 positive cell in Si-ACP/PM treated group was significantly higher


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than that of the control, PM and ACP/PM groups at day 7. In addition, RT-PCR analysis further demonstrated that the expression of angiogenesis related gene (VEGF and TGF-β) in the Si-ACP/PM group were markedly elevated when compared with that of other groups (Fig. 5C and D).

Figure 5. (A) CD31 immunofluorescence staining of the wound tissue treated with different scaffold at 7 days. Green: CD31; Blue: nuclei; The newly formed vessels were marked with red arrows. (B) Quantification analysis of CD31-positive vessels per high-power field (HPF) at 7 days. The gene


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expression of the VEGR (C) and the TGF-β (D) in the control, PM, ACP/PM and Si-ACP/PM groups. (*P < 0.05, **P< 0.01, ***P< 0.001) The Si-ACP/PM scaffolds stimulating the re-epithelialization and ECM formation in diabetic wounds. Masson’s trichrome staining was performed to investigate the epidermal migration and the collagen deposition in the wound beds of the four groups (control, PM, ACP/PM and Si-ACP/PM) in vivo. Compared with the control, PM and ACP/PM treated groups (Fig. 6A1-C1), the length of the new epithelia in the Si-ACP/PM treated group was significantly increased and a continuous and uniform epithelium could be observed after 15 days (Fig. 6D1). The high-magnification optical images in Fig. 6A2-D2 showed that, compared with the control, PM and ACP/PM groups, there were more collagen fibers with extensive and orderly-arranged structures observed in the new healed wound site of the Si-ACP/PM treated group (Fig. 6D2).


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Figure 6. Masson’s trichrome staining images of the wound tissue sections of the control, PM, ACP/PM and Si-ACP/PM treated groups at 15 days, revealing the re-epithelialization (A1-D1) and collagen deposition (A2-D2) in diabetic wound beds in the three groups. 4. Discussion Diabetic chronic wound brings not only heavy health burden to patients, but also economic challenge to the society, which has drawn worldwide attention. It has been widely accepted that angiogenesis play an important role in wound healing. However, how to stimulate angiogenesis in the wound region is still a challenge.4 In this study, a spaced-oriented electrospun membrane (PM) with Si-CAP nanocoating was successfully prepared via combining the


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patterning electrospinning with PLD technology to stimulate angiogenesis in wound site and thereby accelerate diabetic wound healing. Recently, patterned electrospun membranes have been widely used in tissue engineering13, 36-37. Compared with randomly deposited fiber scaffolds, the different regions of patterned scaffold show altered nanofiber density, and nanofibers deposited between the embossments exhibited a higher porosity with large pore structures, which is beneficial for oxygen and nutrients transportation and can support cell ingrowth.36,

38

Beyond that, numerous

researched have demonstrated that the surface topography of scaffolds play important roles in cells differentiation.8-10 Previous studies showed that cells cultured on the patterned scaffold would be great affected by the micro/nano topographic cues of the scaffold, and lead to the large shape change of cells cytoskeleton and nuclei, which play a positive roles in the differentiation of endothelial cells.13-14, 35 Based on the previous study, herein, we chose a wire spring as collector and in the process of electrospinning, the nanofibers deposited randomly with a high density on the embossments, while others loosely aligned into a uniaxial array between the embossments to form the spaced-oriented microstructure. This kind of microstructure exhibited multiple superiorities in structures. First, the embossments provided sufficient mechanical strength to maintain the structural integrity of the scaffold. Second, the nanofibers sparsely deposited between the embossments leading to a loose fibrous structure, which could enhance the transportation of oxygen and


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nutrients to cells and supported cell migration. In addition, those fibers between the embossments exhibited an aligned structure, which had been proved to stimulate the angiogenesis of HUVECs.14 To further improve the bioactivity of the scaffold, uniform silicon doped calcium phosphate (Si-ACP) particles with the diameter of 40-50 nm were coated on the PM by using PLD technique. SEM images revealed that the prepared ACP nanocoating showed well distribution on the surface of the composite scaffolds (Fig. 1). It has been demonstrated that the Ca ion can enhance the proliferation of fibroblast cell, resulting in the formation of epidermis and collagen deposition on the dermis.26-27,

39

Previous studies

have also proved that the hydrophilic surface benefits the cell adhesion and the cell growth.35 In this study, after coated with the inorganic particles, the hydrophilic of scaffolds was significantly improved (Fig. 1A4-C4 ）and the cells cultured both on the ACP/PM and Si-ACP/PM scaffolds showed improved proliferation behaviors (Fig. 3C). Current study has confirmed that silicon as an essential element in our body plays a significant role in cell behaviors, including stimulating the proliferation and secretion of angiogenic growth factors of fibroblasts as well as endothelial cells.20, 40-42 In according with the previous studies, the number of cells cultured on the composite Si-ACP/PM was higher than that of ACP/PM scaffold, which implied that the Si ion released from the Si-ACP/PM group (0.523 μg/mL - 2.13 μg/mL) was contributed to the improved cell proliferation.29,

43-44


The results of transwell

24

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invasion assay in vitro showed the number of the migration HUVECs in the Si-ACP/PM group was larger than other groups. The tube forming assay also revealed that the Si ions released from the Si-ACP/PM membranes resulted in the formation of the capillary-like structure with longer tubes and more nodes in the capillary-like structure, which indicated the higher pro-angiogenic ability of HUVECs than other groups. Plenty of studies have reported that the proliferation of endothelial cells could be effectively stimulated with suitable concentration of Si ions, while Si ion with a high concentration may be toxic and suppress the proliferation of HUVECs.29 In particularly, we also fabricated the Si-ACP scaffolds with different amount of doped silicon (4.8Si-ACP/PM and 9.6Si-ACP/PM) and evaluated the cells growth behaviors on those scaffolds (as shown in Fig. S3 and Fig. S4). The results showed that the concentrations of Si ions released from 4.8Si-ACP/PM and 9.6Si-ACP/PM at 7 days were about 3.231 μg/mL and 4.132 μg/mL (Fig. S3), respectively, which were significantly exceed optimum range (0.6-1.7 μg/mL) as reported in the previous studies.20,

29

The in vitro studies further revealed that, after

cultured for 7 days , the number of cells cultured in both the 4.8Si-ACP/PM and 9.6Si-ACP/PM groups were significantly decreased compared with that of the 2.4Si-ACP/PM group. The migration assay (Fig. S4B and D) and tuber form assays (Fig. S4C, E and F) had also demonstrated that, both the 4.8Si-ACP/PM and 9.6Si-ACP/PM scaffolds shown obviously inhibition on the migration and tube formation capacities of HUVECs. These results confirmed


25


Page 26 of 44

the fact that the amount of doped Si ions have a great influence on the biological performance of scaffolds and the Si-ACP/PM scaffolds, in which the amount of doped Si was 2.4%, displayed the optimal biological activity. Consist with the in vitro results, the in vivo diabetic wound healing assay showed that the Si-ACP/PM scaffolds exhibited the highest healing efficiency of diabetic wound when compared to the control, PM and ACP/PM groups. As is well known, the cell microenvironment at the diabetic wound site is interrupted by the sustaining high glucose level, which leads to poor angiogenesis and chronic inflammation.4,

45

The lack of angiogenesis is

considered as one of the main causes to the persistence of chronic wounds.44 To

investigate

the

early

newly-formed

vessels

in

wound

beds,

immunofluorescence staining of CD31 was performed after 7 days post-surgery. The results showed that the expression of CD31 was significantly enhanced in the Si-ACP/PM group as compared with that of the control, PM and ACP/PM groups (Fig. 5B). The main possible reasons for the improved the pro-angiogenesis was that the cells seeded on the spaced-oriented scaffold would experience a large degree of elongation and the angiogenesis ability of HUVCEs was significantly improved.14 Furthermore, the Si ions released from the Si-ACP nanocoating would promote the expression of HIF-1α and VEGF.46 The result of RT-PCR assays further showed that, the expression of the angiogenesis-related gene, including VEGF and bFGF, was significantly upregulated in the Si-ACP/PM group,


26

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which confirmed the stimulatory effect of Si-ACP/PM scaffolds on the angiogenesis differentiation of wound bed in vivo. Once the angiogenesis in the wound sites is promoted the blood capillaries could deliver oxygen and nutrients into the new tissue formation site to accelerate the granulation tissue formation and collagen deposition.47 The results of Masson’s trichrome staining demonstrated that the length and the thickness of the new epithelia were significantly increased in the Si-ACP/PM group and the wound was almost epithelized. Accordingly, the deposition of collagen fibers was obviously increased in the Si-ACP/PM group. Moreover, the collagen fibers observed in the same group were much more intensive and ordered than that of other groups at 15 days.48 5. Conclusion In this study, we successfully prepared a space-oriented nanofibrous electrospun scaffold with Si-ACP nanocoating as an ideal wound dressing for accelerating diabetic wound healing by the combination of the patterning electrospinning and PLD technique. Our results indicated that most of nanofibers in the scaffolds were randomly deposited to form columnar embossments, while others loosely aligned between embossments. The silicon doped ACP nanoparticles were well distributed on the surface of the nanofibers and the Si ions could sustain release from the nanocoatings. The

in vitro study demonstrated that the space-oriented structures and the silicon ions released from the scaffolds could synergistically promote the


27


Page 28 of 44

proliferation, migration and differentiation of the HUVECs. The in vivo study further proved that the prepared Si-ACP/PM scaffold markedly improved angiogenesis, re-epithelialization and collagen deposition in the wound site, which ultimately accelerate the progress of the diabetic wound healing. Our results indicate that this kind of composite scaffold is a promising bioactive material for diabetic wound healing application. Acknowledgments



This work was supported by the National Natural Science Foundation of China (Nos. 81501597). AUTHOR INFORMATION



Corresponding Authors H.

Xu:

[email protected],

Q.

Ke:

[email protected],

Z.

Yi:

[email protected] ORCID He Xu: 0000-0002-8345-7203 Notes The authors declare no competing financial interest. 

ASSOCIATED CONTENT Supporting Information


28

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SEM, EDS, and XRD of xSi-HAp. Morphology, water contact angle and Si ions release profiles of 4.8 and 9.6Si-ACP/PM. Transwell

assay

and

tube

forming of the HUVECs in 4.8 and 9.6Si-ACP/PM. Cell proliferation of HUVECs and HAF cultured on xSi-ACP/PM 

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Graphical Abstract


37


Figure 1. Optical images (A1-C1), SEM images (A2-C2), the high-magnification SEM images (A3-C3) and water contact angles (A4-C4) of the composite scaffold: patterned membrane (PM) (A1-A4), PM coated with ACP (B1-B3) and PM coated with Si-ACP (C1-C3).


Page 38 of 44

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Figure. 2 Si ions release profiles of Si-ACP/PM



Figure 3. (A) The transwell assay of HUVECs co-cultured with different scaffolds after 12 hours and (D) the corresponding quantitative analysis of migrated cells. (B) The tube formation assay of HUVECs co-cultured with different scaffolds after 6 hours of culture. (C) Proliferation of HUVECs after 1, 3 and 7 days of culture with different scaffolds. Quantitation of the tube length (E) and number of nodes (F) formed after 6 h of culture in the tube formation assay, respectively. (*P < 0.05, **P< 0.01, ***P< 0.001)


Page 40 of 44

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Figure 4. (A) Representative images of the wound with different treatments group (control, PM, ACP/PM and Si-ACP/PM) at 0, 5, 7, 9 and 13 days post-surgery. (B) The trace of wound region from day 0 (blue) to days n (yellow). (C) Statistical analysis of the area of wound area in each group at day 0, 5, 7, 9 and 13 days post-surgery. (*P < 0.05, **P< 0.01, ***P< 0.001)



Figure 5. (A) CD31 immunofluorescence staining of the wound tissue treated with different scaffold at 7 days. Green: CD31; Blue: nuclei; The newly formed vessels were marked with red arrows. (B) Quantification analysis of CD31-positive vessels per high-power field (HPF) at 7 days. The gene expression of the VEGR (C) and the TGF-β (D) in the control, PM, ACP/PM and Si-ACP/PM groups. (*P < 0.05, **P< 0.01, ***P< 0.001)


Page 42 of 44

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Figure 6. Masson’s trichrome staining images of the wound tissue sections of the control, PM, ACP/PM and Si-ACP/PM treated groups at 15 days, revealing the re-epithelialization (A1-D1) and collagen deposition (A2D2) in diabetic wound beds in the three groups.



Graphical Abstract


Page 44 of 44

Space-Oriented Nanofibrous Scaffold with Silicon-Doped Amorphous

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