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Back to Journal »International Journal of Nanomedicine» Volume 13

Author Griffin M, Palgrave R, Baldovino-Medrano VG, Butler PE, Kalaskar DM 

Published on October 8, 2018, the 2018 volume: 13 pages 6123-6141

DOI https://doi.org/10.2147/IJN.S167637

Single anonymous peer review

Editor approved for publication: Dr. Thomas J Webster

Michelle Griffin,1–3 Robert Palgrave,4 Víctor G Baldovino-Medrano,5 Peter E Butler,1–3 Deepak M Kalaskar1,6 1UCL Center for Nanotechnology and Regenerative Medicine, Department of Surgery and Interventional Sciences, University College London, London, UK ; 2Royal Free London NHS Foundation Trust Hospital, London, UK; 3Charles Wolfson Reconstructive Surgery Center, Royal Free London NHS Foundation Trust Hospital, London, UK; 4Department of Chemistry, University College London, UK; 5Surface Science Laboratory ( SurfLab), School of Chemical Engineering, Pied Cuesta, Colombia; 6UCL Institute of Orthopaedics and Musculoskeletal Sciences, Department of Surgery and Interventional Sciences, University College London, London, UK Background: Tissue integration and vascularization are successfully implanted for An important standard for synthetic biomaterials implanted subcutaneously. Goal: We report using argon (Ar), oxygen (O2), and nitrogen (N2) using polyurethane polymers to optimize plasma surface modification (PSM) to enhance tissue integration and angiogenesis. Method: Compare the volume and surface characteristics of the stent before and after using PSM with Ar, O2, and N2. The viability and adhesion of human dermal fibroblasts (HDF) on the modified scaffold were compared. The extracellular matrix formed by HDF on the modified scaffold was evaluated. The scaffold was implanted subcutaneously in a mouse model for 3 months to analyze tissue integration, angiogenesis, and capsule formation. Result: Surface analysis shows that the interface modification (chemistry, morphology and wettability) achieved by PSM is unique and varies according to the gas used. O2 plasma caused extensive changes in interface properties, while Ar treatment caused moderate changes. The N2 plasma has the least effect on the surface chemistry of the polymer. The PSM-treated stent significantly enhanced the activity and growth of HDF within 21 days (P <0.05). Among all three gases, the Ar modification showed the highest protein adsorption. The Ar-modified scaffold also showed adhesion-related proteins (vinculin, focal adhesion kinase, talin and paxillin; P<0.05) and extracellular matrix marker genes (type I collagen, fibronectin, laminin and elastin ) And associated protein deposition is significantly upregulated by HDF. Compared with the O2 and N2 modified stents, the Ar modified stent showed the highest tissue integration and angiogenesis and the lowest capsule formation after subcutaneous implantation after 3 months. Conclusion: PSM using Ar is a cost-effective method to improve tissue integration and angiogenesis of subcutaneous implants. Keywords: tissue integration, angiogenesis, surface modification, biomaterials, implants errata for this article have been published

Subcutaneous synthetic implants are used in a variety of surgical procedures to restore anatomical structures after surgical resection, including reconstruction of the breast, face, abdomen, and pelvis. However, the infection and squeezing of the synthetic material under the skin has reached unacceptable levels, leading to a significant increase in the incidence of patients and placing demands on medical and healthcare resources. One of the main reasons for the poor performance of synthetic materials is poor integration with surrounding subcutaneous tissues and lack of angiogenesis, leading to lack of anchorage, which leads to implant infection and compression. 1-3

In order to improve the results of synthetic biomaterials, the matrix should be designed to guide the desired cellular response by mimicking the extracellular matrix (ECM). With the latest advances in nanotechnology, nanocomposite materials provide a matrix whose size mimics natural ECM and improve their biocompatibility. 4-8 Our group is currently developing a subcutaneous implant using nano-composite polyurethane. 4,5 This polyurethane polymer has been extensively tested and meets international standards for biocompatibility (ISO 10993). Due to its hydrophobicity, it has been extensively studied for hollow organs, including vascular grafts 9 and lacrimal ducts 10, where fluid flow is the main requirement rather than tissue integration.

Various methods have been studied to modify the tissue integration of synthetic materials, including the deposition of proteins, growth factors, and chemical groups. 9-16 The creation of functional materials is essential to change the biological processes that occur at the material interface because they are determined by the physical and chemical properties of the material surface. It is worth noting that manipulating the surface chemistry and wettability of biomaterials is the easiest way to control surface interactions and improve biocompatibility. 17,18 Biomolecules, including proteins and growth factors, can simply be adsorbed to the surface of the material or covalently attached to the surface through a chemical reaction. 19 Many research groups have shown that the adsorption of ECM components to the surface of materials can guide cell behavior, including fibronectin, laminin, collagen, and elastin. 19 Several binding motifs from this type of ECM have been identified and made into short linear amino acid sequences including the RGD motif to improve cell attachment to the surface of biological materials. 19 However, using covalent bonding or adsorption techniques, it is difficult to understand the correct presentation of peptides or proteins and obtain sufficient binding to guide cell behavior. 20 The delivery of growth factors to guide cell behavior has also been demonstrated. Report the s in the scaffold during the manufacturing process by capturing biomolecules. 20 The molecular release kinetics can be controlled by the porosity of the scaffold. 20 However, due to long-term physical retention, growth factors usually lose their biological activity. 20 Electrostatic bonding is another advantageous technique because it mimics the natural interaction of growth factors and ECM. 20 However, the biomolecules presented through this technique may not always be consistent, and therefore may not effectively affect downstream pathways. 20 Overcoming the challenges of using molecules to create a biometric surface, the surface chemistry of biomaterials can be changed to direct cell behavior. 21 Several chemical groups have been shown to affect cell behavior, including carboxyl (-COOH), amine (-NH2), methyl (-CH3) and hydroxyl (-OH) 21 Research shows that -NH2 can improve adhesion and growth And matrix formation. 22,23 It has been shown that carboxyl groups can improve protein adsorption and allow 24 There are several techniques to introduce such chemical groups into the surface of materials, including ion beam implantation and ion beam exchange. 25 However, these technologies require extensive training and high costs. 26 To overcome these challenges, an alternative method of changing the surface chemistry of implants uses plasma surface modification (PSM).

PSM is a cost-effective method to control the physical and chemical properties of the surface of biological materials. 18 PSM can change the wettability of the surface by changing the surface chemistry and morphology. This physiochemical change has been proven to enhance cell adhesion and proliferation, guide cell response and change the blood-biomaterial interface, thereby improving the applicability of the material in clinical use. An important advantage of 27-39 PSM is that the treatment can be used to adjust the surface chemistry and wettability without changing the overall properties of the material.

Radio frequency plasma is the most widely used plasma source, and it involves passing an electric current through a gas. 18 Various gases have been used together with PSM to impart specific surface chemistry or etching effects to change the surface topography, thereby adjusting the surface composition. Although PSM has been used to change the interface properties of materials extensively for many years, there is no comprehensive study documenting the effects of various gaseous plasmas on the surface properties of biological materials, and there is no extensive in vitro and in vivo evaluation to confirm its efficacy. Most of the reported studies used multiple gases, parameters, and materials, so it is difficult to compare the efficiency of various PSM methods. 18

This study aims to compare the three commonly used gases of PSM, including argon (Ar), oxygen (O2) and nitrogen (N2), to optimize the plasma process to enhance the tissue integration and angiogenesis of the polyurethane stent. In this study, we optimized the PSM process by processing the stent for different times to determine the impact on the polyurethane surface and overall performance. The research was subsequently expanded through in vitro and in vivo studies to find the most effective modification process to enhance tissue integration and angiogenesis. The method developed in this research can be easily converted to the surface of other biomaterials, providing a major advancement in the development of materials for surgical applications that require long-term subcutaneous implantation.

The polymer was synthesized using a two-part method as previously described. 11,12 In short, polycarbonate polyol (2000 molecular weight [Mw]) and trans-cyclohexane chloroisobutyl-silses-106 quinoxane (Hybrid Plastics Inc., Hattiesburg, MI, USA) Mix and then dissolve the polyoligomeric silsesquioxane (POSS) cage into the polyol solution at 70°C. Then, 4,4'-methylenebis(phenyl isocyanate) was added to form a prepolymer. After that, dimethylacetamide (DMAC) was then slowly added, and the chain was extended by cooling the solution to 40°C. This led to the synthesis of POSS-modified polycarbonate urea-urethane (PU) in DMAC solution. The stent is manufactured using the salt immersion method as described above. 11,12 Sodium chloride (NaCl) and PU in a weight ratio of 3:7 were used in all experiments. First, pour the NaCl/PU mixture on a steel mold, and then put it in an oven at 65°C for 4-5 hours until all the solvent has evaporated. The cast polymer was then placed in deionized water for 7 days to remove all NaCl to produce a porous scaffold. For cell culture analysis, the scaffold is cut into 16 mm diameter discs for use in 24-well plates.

PSM is performed using a radio frequency plasma generator configured for 40 kHz, 100 W and 0.4 mbar flow rate. PSM is performed by placing the stent in O2, Ar, or N2 gas for 1, 2.5, 5, 7.5, or 10 minutes. The untreated (Con) scaffold was used as a control.

As mentioned earlier, using the DSA 100 instrument (KRUSS, Hamburg, Germany), the static drop method was used to analyze the static water contact angle (n=6). 11,12 After changing the length, measure the static contact angle on six supports. The plasma exposure time is 1 to 10 minutes.

As mentioned earlier, the Instron-5565 tensile 224 tester was used to analyze the mechanical properties of the PSM back stent. 11,12 The average Young's modulus of elasticity is reported after analyzing six independent stents (n=6).

The surface structure and structure of the scaffold, especially the pore size and porosity (n=3), were analyzed by SEM. 11,12 In short, the scaffold was fixed with 2.5% w/v glutaraldehyde/PBS for 48 hours. After this, the stent uses CO2 for critical point drying. Then before imaging with the FEI Quanta 200F scanning electron microscope, a sc500 (EMScope; Quorum Technologies, Lewes, UK) sputter coater was used to gold-plated the stent.

X-ray photoelectron spectroscopy (XPS) research

A Thermo Scientific K-Alpha spectrometer was used to evaluate the surface chemistry of the treated and untreated stents as described previously (n=3). 11,12 In short, a monochromatic Al Kα X-ray (h=1,486 eV) is focused on a -μm diameter spot on the surface of the 400 stent, defining the analysis area. The analysis depth is at 5-10 nm of photon energy. Investigating the spectra proved the elemental composition of the surface. Then obtain high-resolution spectra of the main core lines of each element present for chemical state identification. Then, use the CasaXPS software to fit the high-resolution spectra to the Gauss-Lorentz peaks to deconvolute different chemical environments. Use CasaXPS software to fit Gauss-Lorentz peaks to deconvolute different chemical environments.

The surface roughness of the stent was evaluated using AFM (TAP150A) in tapping mode (spring constant 2.919 n/m) as described above (n=3). 11,12 In short, the root mean square roughness was scanned by 5 μm in three areas using NanoScope® analysis software version 1.40 (Bruker Corporation, Billerica, MA, USA). PeakForce quantitative nanomechanical mapping is used to obtain FV modulus using an improved Hertz model.

The average molecular weight and polymer dispersion are determined by GPC analysis. The stent was prepared at a concentration of 1 mg/mL and passed through a 0.22 μm nylon filter before measurement. GPC measurement was performed on an Agilent 1260 infinity system equipped with 2 PLgel 5 μm mixed D columns (300×7.5 mm), a PLgel 5 mm guard column (50×7.5 mm), differential refractive index (DRI) and variable wavelength detector. The temperature of the column and DRI is maintained at 50°C. The system was eluted with DMF containing 5 mM ammonium tetrafluoroborate at a flow rate of 1 mL/min, and the DRI detector was calibrated with a linear narrow poly(methyl methacrylate) standard (n=3) with a narrow molecular weight distribution.

The glass transition temperature (Tg) of the polymer was measured using Q2000 DSC (TA Instruments Ltd., Elstree, UK) at 5 and 10 minutes after PSM treatment. Weigh the stand (5-10 mg) in a sealed Tzero aluminum pan. An empty plate that matches the weight of the bracket plate is used as a reference. The stent is heated from 0°C to 250°C at a rate of 20°C/min. Use Universal Analysis 2000 to analyze the data and use N2 as the purge gas. Use Origin Pro 9.1 software (n=3; OrginLab, Northampton, MA, USA) to draw DSC thermogram.

As mentioned earlier (n=6), the bicinchoninic acid assay was used to determine the amount of total serum protein adsorbed on the unmodified and modified scaffolds (n=6). 11,12

The function of the cell-binding domain of adsorbed fibronectin and vitronectin is presented

The accessibility of the cell-binding domains of bFN and bVN adsorbed on the scaffold was checked by ELISA using monoclonal antibodies (mAbs) against epitopes containing RGD domains, as described by Barrias et al. 16 In short, the scaffold is a protein solution (bFN or bVN) of different concentrations at 37°C. After washing with PBS, all surfaces were blocked with 1% w/v BSA/PBS, and then combined with specific mouse anti-bovine mAb at predetermined optimal concentrations (0.17 μg/mL antibFN and 1 μg/mL anti-bVN in 1 % w /v BSA/PBS, Alpha Laboratories, Eastleigh, UK) Place it at 37°C for 1 hour. After washing 3 times in PBS, the scaffold was conjugated with horseradish peroxidase-conjugated anti-mouse IgG (H+L) secondary antibody (1:1,000 in 1% w/v BSA/PBS; Fisher Scientific, Loughborough , UK) Incubate for 1 hour at 37°C. After further washing, the scaffold was incubated in o-phenylenediamine dihydrochloride substrate (0.4 mg/mL 0.05 M phosphate-citrate buffer, pH 5.0, containing 0.012% v/v hydrogen peroxide). Then use a spectrophotometer to read the OD of the supernatant at 450 nm with reference to 620 nm.

As described by Kalaskar et al., the role of specific integrins in the adhesion of fibroblasts to plasma modified scaffolds was studied. Blocking antibodies α5β1 and α5β3 (Abcam, Cambridge, UK) in cell culture medium at 37°C at a concentration of 1 to 10 mg/mL. Cells (treated with and without blocking antibodies) were seeded on scaffolds treated with different PSMs at a density of 50,000 cells/cm3. After 3 hours, the number of adherent cells was counted, and cell adhesion was calculated by counting the number of adherent cells in three different random fields of three cultures.

Cell culture and cell seeding

As mentioned earlier, the HDF obtained from the European Accredited Cell Culture Collection (ECACC) is stored in Dulbecco's Modified Eagle's medium containing 10% fetal bovine serum and 1% antibiotic solution (Sigma-Aldrich, St Louis, MO, USA) . 11,12 For in vitro experiments, a 16 mm scaffold was placed in a 24-well plate and cultured in complete medium for 24 hours before cell seeding. Each scaffold was seeded with 1×104 cells/cm2. Change the medium every three days.

In order to study the structure of HDF seeded on the scaffold, the cytoskeleton was stained with actin phalloidin 24 hours later. 11,12 Use Alamar Blue™ Assay (Sigma-Aldrich) and Fluorescent Hoechst DNA Quantification Kit (Sigma-Aldrich) to evaluate the activity of HDF. Check the DNA content on Days 1, 2, 4, 7, 14 and 21, as described above Description (n=6). 11,12

The real-time quantitative polymerase chain reaction (RT-qPCR) was performed as previously described. 40 First, RNA was extracted using Tri-Reagent (Life Technologies, Waltham, MA, USA) on the 7th and 14th days. Second, reverse transcription of RNA using Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI, USA). After that, RT-qPCR was completed using ABI Prism 7500 Sequence Detection System (Thermo Fisher Scientific, Waltham, MA, USA) and QuantiTect SYBR Green PCR Kit (Qiagen, Hilden, Germany). GAPDH was used as a housekeeping gene to normalize the data using the 2-ΔΔCt method. The primers used in the study are shown in Table 1.

Table 1 The RT-qPCR primer sequence used in this study is abbreviated as: RT-PCR, real-time polymerase chain reaction.

The expression of ECM and adhesion-related proteins uses the method described above. 40 On the 14th day, the medium was removed and the scaffold was washed and fixed in 4% paraformaldehyde. After incubating overnight, the scaffold was further washed. After permeabilization (0.5% Triton X-100), the stent was blocked (0.5% BSA). After overnight incubation with the primary antibody at 4°C, further washes (488 Alexa secondary antibody, 1:500) before incubating the scaffold in the secondary antibody for 2 hours. Finally, the nuclei were stained with Hoechst 33258 (2.5 μg/mL final concentration). The antibodies used in immunocytochemistry in this study are shown in Table 2.

Table 2 Details of the primary antibodies used in this study

Enzyme-linked immunosorbent assay (ELISA) for vascular endothelial growth factor (VEGF)

According to the manufacturer's instructions, ELISA analysis (Quantikine; R&D Systems, Inc., Minneapolis, MN, USA) was used to evaluate the VEGF secretion of HDF. On days 4, 7, 10, and 14, the cell culture supernatant was removed and evaluated using ELISA as previously described. 41

After PSM treatment, two 4 mm (diameter) × 1 mm (thickness) discs were subcutaneously implanted into a 4-month-old BALB/c mouse (Charles River Laboratory, Wilmington, Massachusetts, USA) The back was subcutaneously (n=6).11 The intervertebral disc was implanted through a small incision and sealed with intermittent 5,0 monocryl. All experiments were approved by the animal care committee of the local government and were performed in accordance with the animal welfare regulations of the United Kingdom. In the 6th and 12th weeks, CO2 was used to asphyxiate, the animals were sacrificed, the stent was removed and fixed in 4% paraformaldehyde. Excise the stent with approximately 0.5 cm around the round dermis. Then the fixed stent was embedded in paraffin and cut into 3 μm sections for histological analysis. H&E staining was performed according to standard procedures to evaluate tissue integration and CD31 immunohistochemical staining for endothelial cell detection, as in previous studies. 11 The study was approved and conducted in accordance with the Animal Research Guidelines of the Department of the Home Office of the United Kingdom.

GraphPad (Prism) is used for statistical analysis using one-way or two-way analysis of variance and Tukey HSD post-hoc test. P value<0.05 was considered statistically significant.

The untreated stent (Con) exhibited a static water contact angle of 67°±7°. Compared with the unmodified stent, the contact angle of the stent was significantly reduced after being treated with Ar (32.2°±2°), N2 (62.7°±4°) and O2 (14.8°±7°) for 1 minute (P< 0.05) (Con; Figure 1A).

Figure 1 The effect of Ar, N2 and O2 plasma treatment on (A) wettability (static water contact angle), (B) surface roughness and (C) surface elastic modulus of polyurethane stents. Note: The change in surface properties is presented as a function of processing time. For plasma treatment times of different durations, the normalized element composition measured by XPS analysis is expressed as (D) carbon content ratio, (E) nitrogen content ratio, and (F) oxygen content ratio. Abbreviations: Ar, argon; scam, untreated; N2, nitrogen; O2, oxygen; XPS, X-ray photoelectron spectroscopy.

PSM affects the surface roughness of the stent after Ar, O2 and N2 treatment. The roughness of the unmodified stent (Con) is 8±1 nm (Figure 1B). After 5 minutes of treatment, the changes in surface roughness after Ar and N2 were only significant compared with the control (N2, 1 minute, 9±1 nm; N2, 5 minutes, 13±2 nm; Ar, 1 minute, 9± 1 nm; Ar, 5 minutes, 11±2 nm). However, O2 plasma causes the surface roughness to increase linearly with the treatment time (O2, 1 minute, 16±3 nm; O2, 10 minutes, 38±4 nm), indicating that it is the most radical plasma treatment. This change results in Confirm the use of SEM (Figure S1).

Surface elastic modulus using AFM

Compared with the control, with the increase of treatment time, Ar and N2 plasma treatment showed no change in surface elastic modulus (Con; Figure 1C). However, with O2 treatment, the surface elastic modulus increased linearly with the increase of treatment time (P<0.05).

For XPS analysis, the relative molar concentrations of C, N, and O from all stents were normalized and shown in Figure 1D-F. After treatment with Ar and O2, the C/(N+O) ratio showed a gradual decrease. Some people believe that this trend may be related to the removal of residual carbon contaminants from the surface (Figure 1D). In contrast, after N2 treatment, the C/(N+O) ratio did not follow any clear pattern. Observed trends indicate that under these conditions, cyclic removal and deposition of carbon will occur on the surface of the material.

On the other hand, after treatment with all three gases, the N/(C+O) ratio showed only a small change (Figure 1E), indicating that the surface N concentration of all stents did not change significantly. For the O/(C+N) ratio, a significant increase was observed after one minute during O2 treatment (Figure 1F). Further stent treatment did not increase the O/(C+N) ratio. For Ar treatment, the O/(C+N) ratio is gradually increased up to 5 minutes and remains unchanged even after a longer treatment time. N2 treatment does not follow any specific trend. The calculated ratio showed similarity to the pattern observed with C/(N+O) treated with N2 plasma. In general, the C ratio decreases with Ar and O2 treatment, and in response, the O ratio increases with Ar and O2 treatment. On the other hand, during the treatment process, N2 treatment does not seem to have a significant effect on polymer chemistry.

Volume characteristics, including mechanical testing, DSC analysis, and molecular weight analysis

The changes in the overall performance of the stent after PSM were checked by GPC, DSC and mechanical properties. After 5 minutes of treatment, no matter what the gas type, GPC did not find a significant change in the average molecular weight (Mn) or average molecular weight (Mw) (Figure S2). After being modified by plasma treatment, compared with the untreated stent, there is no difference in the Young's modulus of the stent using Ar, N2 or O2 (Con; Figure S3). After using different PSM techniques analyzed by DSC, Tg did not change significantly (Figure S4).

For all gaseous plasmas, 5 minutes provided the highest cell attachment (Figure 2A). However, among all the test gases at 5 minutes, compared with the O2 and N2 modified scaffolds, the Ar modified scaffolds observed the highest cell adhesion at 24 hours (P<0.05).

Figure 2 HDF adhered to the stent treated with plasma surface modification after 24 hours. Note: (A) DNA detection with different treatment time using Ar, N2 and O2 plasma treatment showed that the cell attachment level was the highest after 5 minutes of PSM treatment. (B) The effect of plasma modification on actin cytoskeleton organization and vinculin expression shows that cells spread on all scaffolds. There is evidence that FAK (white arrow) is formed after 24 hours (green: vinculin staining; red: actin) Silk and blue: DAPI is used for nuclear staining). (C) Quantitative expression of adhesion-related genes, vinculin, FAK, talin, and paxillin showed that after 5 minutes of treatment, the expression of Ar-modified stents was significantly higher than that of other plasma gas and untreated stents. Up-regulation (*P<0.05). The fold change represents the difference compared to the housekeeping gene GAPDH of cells grown on unmodified scaffolds (Con). Abbreviations: Ar, argon; scam, untreated; FAK, focal adhesion kinase; HDF, human dermal fibroblasts; N2, nitrogen; O2, oxygen; TCP, tissue culture plate; Neg con, negative control with primary antibody omitted.

When the cell morphology was checked by staining the cytoskeleton of the cells, HDF cells showed proliferation on all plasma modified scaffolds and unmodified scaffolds (Figure 2B). In addition, the expression of HDF vinculin was confirmed on all scaffolds by immunocytochemistry after 24 hours. Compared with the unmodified scaffold, the expression of vinculin, focal adhesion kinase (FAK), talin and paxillin was higher on the PSM scaffold (P<0.05). In the tested gases, the expression of adhesion-related genes on Ar was significantly higher than that of the unmodified N2 and O2 scaffolds (P <0.05) (Figure 2C).

The short-term cell viability determined using Alamar Blue assay and DNA content analysis was significantly higher on the plasma modified scaffold than the unmodified control scaffold with all processing gases (P <0.05; Figure 3). For all treatment gases, the maximum level of cell growth was observed after 5 minutes of treatment compared to other treatment times.

Figure 3 Human dermal fibroblast viability (A) and DNA (B) of the scaffold treated with plasma surface modification within 21 days. Note: Compared with the untreated scaffold (Con), for Ar, N2 and O2 plasma treated for different time (n=6), the viability was measured using Alamar Blue and DNA assays, respectively. Abbreviations: Ar, argon; scam, untreated; N2, nitrogen; O2, oxygen.

The type, quantity and conformation of essential proteins at the surface interface determine cell adhesion, attachment and long-term growth. To understand the increase in HDF cell growth on the PSM scaffold, the total protein adsorption on all scaffolds was quantified (Figure 4A). Compared with the unmodified scaffold, the PSM scaffold showed significantly higher total protein adsorption. Among the various treatment times used, plasma treatment with all test gases for 5 minutes resulted in the highest protein adsorption from the serum-containing medium compared to the control. In addition, when comparing various test gases, Ar (5 minutes) showed the highest protein adsorption compared to N2 and O2. To analyze the role of specific proteins in cell adhesion, the adsorption of the most common ECM proteins, FN and VN, was quantified (Figure S5). It was found that the adsorption of FN on the scaffolds treated with N2 and O2 and the tissue culture plate (TCP; Figure S5) was the highest, reaching saturation after 5 μg/mL, while the adsorption of VN on Ar was the highest with TCP (Figure S5). Blocking the integrin receptors of VN and FN indicates that fibroblasts use different receptors to adhere to the modified surface (Figure 4B and C). Fibroblasts use fibronectin receptor α5β1 to adhere to N2 and O2 modified surfaces, but use vitronectin receptor αVβ3 on Ar modified surfaces. Since the 5-minute treatment showed the highest protein adsorption, followed by the cell viability and growth of all gases, this treatment time was further used to understand the mechanism of cell interaction, including the role of specific protein adsorption and the interaction of cell surface receptors And their effects on tissue integration and angiogenesis.

Figure 4 Protein adsorption study. (A) The total protein adsorption on the scaffold after plasma surface modification. The BCA assay was used to assess total serum protein adsorption. For all test gases, after 5 minutes of PSM, the total serum protein adsorption on all stents was significantly higher (****P<0.0001). Ar showed the highest protein adsorption of all scaffolds (n = 6). (B) Fibronectin and (C) Vitronectin integrin blocking experiments to evaluate the specificity of adsorbed proteins on the scaffold treated with plasma surface modification. Note: Blocking fibronectin receptor α5β1 with fibronectin blocking antibody (Anti-FN) resulted in a dose-dependent decrease in cell adhesion on the stent treated with N2 and O2, but not on the stent treated with Ar, confirming The fibronectin-mediated effect is attached to the scaffold treated with N2 and O2. The use of vitronectin blocking antibody (Anti-VN) to block the vitronectin receptor αVβ3 resulted in a dose-dependent decrease in cell adhesion to the stent treated with Ar, but not on the stent treated with N2 and O2, confirming vitronectin The main effect of protein-mediated attachment is on the Ar scaffold (n=6). Abbreviations: Ar, argon; BCA, bicinchoninic acid; scam, untreated; N2, nitrogen; O2, oxygen; PSM, plasma surface modification; TCP, tissue culture plate.

Compared with the N2 and O2 modified scaffolds, the mRNA expression and release of ECM factors of type I collagen, elastin, laminin and fibronectin were higher on the Ar modified scaffold (P<0.05; Figure 5). There is no difference in the expression of type III collagen by HDF between the scaffolds (Figure 5). HDF's protein expression of collagen, elastin, and fibronectin was confirmed on the scaffold using immunocytochemistry after using PSM of all gases and on the unmodified scaffold (Figure S6).

Figure 5 Gene expression of ECM markers in human dermal fibroblasts on the plasma modified scaffold using RT-qPCR. Note: Compared with other scaffolds, 14 days after gene expression was confirmed, the ECM markers on the Ar modified scaffold were significantly up-regulated (*P<0.05). All stents were treated with corresponding plasma surface modification for 5 minutes. The fold change represents the difference compared to the housekeeping gene GAPDH of cells grown on unmodified scaffolds (Con). *P<0.05. Abbreviations: Ar, argon; Col I, type I collagen; Col III, type III collagen; scam, untreated; ECM, extracellular matrix; FN, fibronectin; Lm, laminin; N2, nitrogen; O2 , Oxygen; RT-qPCR, real-time quantitative polymerase chain reaction.

Evaluation of tissue integration and angiogenesis in vivo

In order to evaluate the ability of the stent to support tissue integration in vivo, the stent was implanted subcutaneously in a mouse model for 6 and 12 weeks, and the levels of tissue integration and angiogenesis were compared. Figure 6A shows the histology and immunohistochemistry of explanted scaffolds treated with three different gases and unmodified scaffolds. Quantitative H&E staining confirmed that at 6 weeks and 12 weeks, compared with O2, N2 treatment and unmodified stents, the level of tissue ingrowth of the Ar-modified scaffold was significantly higher (P<0.05; Figure 6B). After 12 weeks, the degree of capsule formation was also measured, and it was found that the degree of capsule formation after PSM was significantly reduced compared with the unmodified stent (Figure S7). CD31 staining was used to assess vascularization in vivo to identify capillaries that grow into the stent after different PSMs (Figure 7A). The analysis showed that Ar-modified stents had greater blood vessel formation than other plasma-modified stents and unmodified stents (P <0.05; Figure 7B). In addition, compared with N2 and O2 modified stents, the secretion and expression of angiogenic growth factor, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor on Ar-modified stents were also higher in vitro (Figure 7C). ).

Figure 6 The tissue integration of the plasma modified scaffold in the subcutaneously implanted mouse model for 6 weeks and 12 weeks. Note: (A) Compared with the N2 and O2 modified scaffolds and unmodified (Con) scaffolds stained with H&E, the Ar modified scaffolds found increased tissue ingrowth after 6 and 12 weeks. The tissue integration of O2 and N2 modified scaffolds showed similar levels. Scale bar: 300 μm. (B) Quantification shows that Ar has the highest tissue ingrowth compared with other scaffolds. All stents were treated with corresponding plasma surface modification for 5 minutes. *P<0.05, **P<0.01. Abbreviations: Ar, argon; scam, untreated; N2, nitrogen; O2, oxygen.

Figure 7 Angiogenesis of plasma modified stent after five minutes of treatment. Note: (A) Assessment of angiogenesis in the mouse model after 12 weeks. Use the CD31 marker to identify the ingrowth of blood vessels, where the number of capillaries is identified (red arrow). Scale bar: 250 μm. (B) Quantification shows that compared with N2 and O2 modified stents, Ar modified stents have significantly higher capillary counts. (C) Analysis of VEGF and bFGF secreted by human dermal fibroblasts. Left: VEGF secretion measured by ELISA showed that HDF increased protein secretion on Ar-modified scaffolds at 7, 10, and 14 days compared with N2 and O2 modified scaffolds and unmodified scaffolds (Con). Right: Compared with N2 and O2 modified scaffolds, Ar modified scaffolds increased the mRNA expression of VEGF and bFGF at 14 days. The fold change indicates the difference in the expression of the housekeeping gene GAPDH between the cells grown on the unmodified scaffold (Con) and the modified scaffold. *P<0.05, **P<0.01. Abbreviations: Ar, argon; bFGF, basic fibroblast growth factor; scam, untreated; N2, nitrogen; O2, oxygen; VEGF, vascular endothelial growth factor.

Surgical implants fail due to poor integration with surrounding tissues and lack of vascularization. 2 In this study, we used the cell-compatible material polyurethane to modify it with PSM to overcome this challenge. Although the usefulness of PSM has been widely documented, there is a lack of information on the efficiency and optimal process parameters of the various gases used in PSM, which can be used to enhance the tissue integration and angiogenesis of synthetic implants.

From the surface and bulk analysis, it is obvious that the bulk properties of the polymer are not affected by the plasma treatment for different lengths of time, and only the surface and interface properties have changed. This is consistent with the previous report, which indicated that PSM has no effect on the overall performance of the material. 11,42,43 Detailed surface analysis showed that all treatment gases significantly reduced the surface wettability and resulted in the morphology and surface elastic modulus of the PSM scaffold compared with the unmodified scaffold (Figure 1; P < 0.05). As the treatment time increased, the O2 treated stent showed increased roughness and surface elastic modulus. However, on the other hand, N2 and Ar treatments showed very small changes in roughness and surface elastic modulus within 10 minutes. The surface wettability of the stent was reversed by PSM after being treated with Ar, N2, and O2 for 1 minute. After similar Ar and N2 PSM exposure rates, O2 surface modification also showed a greater effect on reducing the contact angle of the stent.

The decrease in contact angle and increase in surface roughness after O2 plasma are consistent with previous reports. 27 Pappa et al. found that O2 plasma significantly reduced the contact angle and increased the surface roughness of the polycaprolactone polymer surface due to the introduction of groups on the surface of the polar polymer. 27 Shah et al. also demonstrated a similar reduction in contact angle after Ar and N2 treatments on the surface of polylactic acid. 30 Due to the deposition of -NH2 chemical functional groups, N2 has been shown to cause hydrophilic surfaces. 36 Due to the deposition of polar functional groups (-OH) groups, Ar has been shown to produce hydrophilic surfaces. 33 In this study, we found that the roughness increased slightly after Ar and N2 plasma treatment for 5 minutes. Previous studies have shown that the surface roughness will increase and decrease after Ar and N2 plasma treatment. 30,32,37,39 The differences in the literature may be due to the different plasma gas intensities generated by the comparative methods in the research. 30

XPS analysis confirmed that Ar and O2 plasma treatment is an effective carbon remover. The decrease in the carbon ratio and the increase in the N2 ratio appear to be proportional to these two processing gases. As the carbon is removed, N2 under the surface of the support may be exposed. It should also be noted that some of the detected carbon may be related to accidental contamination, which is common in XPS analysis. Ar and O2 plasma treatments also showed an increase in O2 deposition. On the other hand, N2 treatment shows a pattern that can prove periodic N2 deposition, carbon removal, and O2 deposition on the surface, indicating that it has little effect on surface chemistry. In addition, this also shows that the N2 plasma has the least influence on the surface chemistry of the stent. Taking into account all the surface analysis results, it can be shown with certainty that among all the three gases tested, the changes brought about by O2 treatment are the most significant, Ar shows moderate changes, and N2 treatment has the least effect on surface properties. These changes may be caused by differences in the reactivity of the plasma gas. 30 Compared with less reactive gases (such as Ar and N2), processing reactive gases such as O2 will bring more surface changes. 30

In terms of mechanical properties, Mw, Mn, and Tg after increasing Ar, O2 and N2 treatments, the overall performance changes after PSM were studied. After 10 minutes of Ar, O2 or N2 treatment, the tensile mechanical properties and Tg of the polymer did not change. However, GPC showed a decrease in Mw and Mn after 10 minutes of O2 modification, indicating that less than 10 minutes should be used when using this processing gas to prevent the degradation of the nanocomposite. The surface elastic modulus increases after increasing O2 treatment, which may be due to the oxidative degradation and repolymerization of the polymer to form a harder interface. 44 Tiganis et al. demonstrated that the Young's modulus of the surface of the sample with a small amount of acrylonitrile butadiene styrene increased. Indentation measurements indicate that polymer degradation causes the polymer to become brittle and fail. 44 However, further analysis and reporting of polymer degradation are needed to explain this observation.

In order to find the best cell culture conditions, HDF cells were cultured on PSM-treated polymers for different treatment times. Interestingly, after 5 minutes of exposure, all PSM treatments enhanced the adhesion of HDF on the scaffold (Figure 2A) and growth (Figure 3A and B). Cells do not directly interact with synthetic materials, but are attached to the protein-absorbing layer on the surface of the material. The protein adsorption on the PSM surface is greater, which is related to the enhanced cell adhesion after modification compared with the unmodified scaffold (Figure 4A). It is speculated that although the protein adsorption on the unmodified scaffold is lower, its conformation is correct, allowing fibroblasts similar to the plasma modified scaffold to diffuse, but the lower protein adsorption results in a lower level of cell attachment. However, when the surface is modified with Ar, O2 or N2, more protein is available, resulting in higher cell attachment and cell spread (Figure 4A). Among the three processing gases, Ar showed the highest protein adsorption and cell attachment. In order to understand the reasons behind this, further analysis of cell-protein interactions was carried out.

Cells recognize adsorbed proteins through integrins, which are connected between the ECM and the actin cytoskeleton. 45,46 Integrins form focal adhesion complexes when cells adhere. This complex contains structural proteins including vinculin, alpha-actin and talin, as well as signaling molecules such as paxillin and FAK. 46,47 We observed that HDF on Ar-modified scaffolds increased the expression of vinculin, paxillin, talin and FAK. Compared with other PSM scaffolds, this may help to observe increased cell adhesion and long-term growth on Ar-modified scaffolds.

Interestingly, this study also found that the adhesion of HDF to the plasma-modified scaffold occurs through different integrins. The integrin blocking experiment showed that when the anti-α5β1 integrin antibody was used, the attachment selectivity of cells on O2 and N2 was reduced. This observation confirms the role of fibronectin on these surfaces. It is well known that fibronectin interacts with cells through its classic α5β1 integrin receptor. 46 However, when anti-αVβ3 integrin antibodies were used, the decrease in cell attachment was only observed on the Ar-treated scaffolds, indicating that vitronectin played a major role in cell attachment on these scaffolds. 47 However, due to the use of anti-αVβ3-integrin antibody on the Ar-treated scaffold, the percentage of cell attachment is reduced by about 40%, so the role of other cell surface receptors cannot be ignored.

The data provided in this study indicate that the PSM scaffold provides the best chemical and morphological interface after 5 minutes of processing. Changes in the surface of the polymer interface promote the optimal concentration and conformation of selective adhesion proteins, thereby enhancing cell attachment, growth, and cell function. Due to the moderate changes in surface hydrophilicity, roughness, and surface chemistry, these interface changes caused by Ar may allow the largest number of proteins to be adsorbed in their optimal conformation. Compared with O2 and N2 treatments, these surface changes result in higher cell attachment, growth, and cell function. Figure S8 schematically explains this concept in more detail.

The observed short-term cell behavior is maintained in the long-term culture. The long-term cell survival and growth of HDF cells also resulted in the up-regulation of several ECM-related genes, including type I collagen, fibronectin, elastin, and laminin (Figure 5). The HDF on the Ar-treated scaffold continued to express the highest level of ECM-related genes and deposited corresponding ECM proteins (Figure S6). This confirms that PSM enhances cell function not only in the short term but also in the long-term in vitro culture. Previous studies using PSM have also shown that other anchorage-dependent cells (such as osteoblasts and chondrocytes) have increased ECM production. 48,49

In order to determine that the observed cell behavior was not affected by in vitro culture, further in vivo studies were performed using a mouse model. The histological analysis of the explanted scaffolds after 6 and 12 weeks showed that the level of tissue integration on the PSM scaffold was significantly higher than that of the untreated scaffold. However, compared with the stents treated with O2 and N2, the stents modified with Ar are the highest. During in vivo studies, fibrous capsule formation around synthetic implants is a common phenomenon. The degree of checking the thickness of the fibrous capsule can be used as a good indicator of tissue material response. The study showed that the thickness of the fibrous capsule formed on the PSM scaffold was significantly reduced compared with the untreated scaffold (Figure S7), which indicates that PSM not only promotes tissue integration, but also minimizes inflammation.

The development of new blood vessels after clinical implantation of materials used to repair facial defects is a limiting factor for all three-dimensional bioengineered structures. Many growth factors are responsible for angiogenesis. Ring et al. showed that using the dorsal skinfold chamber test, dermal substitutes and allogeneic bone implants enhanced their blood vessel formation after Ar/hydrogen and Ar/O2 plasma treatments, respectively. 50,51 In the current study, all plasma treatments resulted in increased angiogenesis compared to untreated stents. Ar treatment showed the highest blood vessel formation, as shown by CD31 staining (Figure 7A and B). During in vitro culture, HDF secreted the highest amount of VEGF (Figure 7C), which may support more influx and proliferation of endothelial cells, and allow larger blood vessels to form in the body.

In summary, as far as we know, this study is the first to study in detail the effects of various gases used in PSM on polymer surfaces and their effects on short- and long-term tissue integration and angiogenesis. PSM significantly affects the surface chemistry of the polyurethane polymer without affecting its overall performance. Based on in vitro and in vivo studies on all test gases (Ar, O2, and N2), it was found that the optimal PSM time for polyurethane polymers is 5 minutes. However, Ar modification is optimal for the polyurethane scaffold in this study to enhance cell integration and angiogenesis. This is due to the unique combination of surface chemistry and topography modification imparted by this processing gas.

Ar PSM is a simple, fast and very effective surface modification method that can improve the tissue integration and angiogenesis of polyurethane implants. Future work will study the effect of Ar surface modification on further clinically approved biomedical materials to evaluate its effectiveness at the clinical bedside.

This work was funded by the Medical Research Council and the Action Medical Research Charity, who provided MG with a clinical scholarship to carry out this work; GN 2339.

MG conducted all experiments and wrote the manuscript. RP conducted XPS experiments and XPS data analysis. VGBM analyzed the XPS data. PEB supervised all experiments and wrote the manuscript. DMK supervises all experiments, analyzes data and writes manuscripts. All authors participated in data analysis, drafting and critically revising the paper, finally approved the version to be published, and agreed to be responsible for all aspects of the work.

The authors report no conflicts of interest in this work.

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Figure S1 SEM images of the stent surface modified with Ar, N2 and O2 using plasma surface modification for different lengths of time. Note: Scale bar: 2 μm. Abbreviations: Ar, argon; scam, untreated; N2, nitrogen; O2, oxygen; SEM, scanning electron microscope.

Figure S2 GPC after 5 minutes and 10 minutes plasma surface modification using Ar, N2, and O2 treatments. Note: (A) The influence of plasma modification on Mw. (B) The effect of plasma modification on Mn. (C) The effect of plasma modification on PDI. No significant changes were found after 5 or 10 minutes of Ar or N2 treatment. Compared with the unmodified stent, 10 minutes of O2 treatment resulted in a significant decrease in Mw and Mn (P<0.05). **P<0.01. Abbreviations: Ar, argon; scam, untreated; GPC, gel permeation chromatography; Mn, number of molecules; Mw, molecular weight; N2, nitrogen; O2, oxygen; PDI, polydispersity index.

Figure S3 The mechanical properties of the stent after plasma surface modification. Note: Plasma surface modification shows no change in the tensile Young's modulus for all exposure times (up to 10 minutes). Abbreviations: Ar, argon; scam, untreated; N2, nitrogen; O2, oxygen.

Figure S4 DSC after 5 minutes and 10 minutes of plasma surface modification with Ar, N2, and O2. Note: This study shows that there is no change in Tg after 5 and 10 minutes of plasma surface modification with Ar, N2, or O2 compared to untreated stents (Con). Abbreviations: Ar, argon; DSC, differential scanning calorimetry; N2, nitrogen; O2, oxygen; Tg, glass transition temperature.

Figure S5 The function of the cell-binding domains of fibronectin and vitronectin adsorbed after plasma surface modification. Using mAB analysis, after 5 minutes of exposure to the plasma surface for 1 hour, total fibronectin (A) and (B) vitronectin were adsorbed onto the scaffold. Note: After 5 minutes of N2 or O2 modification, more fibronectin is absorbed into the scaffold compared with the Ar modified and unmodified scaffolds (Con). Compared with all other scaffolds, the adsorption of vitronectin is the largest after Ar modification for 5 minutes. Abbreviations: Ar, argon; scam, untreated; mAB, monoclonal antibody; N2, nitrogen; O2, oxygen; TCP, tissue culture plate.

Figure S6 ECM formation of human dermal fibroblasts after plasma surface modification. Immunocytochemistry confirmed the expression of ECM markers on plasma modified and untreated scaffolds after 14 days (green: type I collagen, elastin, fibronectin; blue: DAPI). Note: Scale bar: 20 μm. Abbreviations: Ar, argon; scam, untreated; ECM, extracellular matrix; N2, nitrogen; Neg con, negative control with primary antibody omitted; O2, oxygen; TCP, tissue culture plate.

Figure S7 After 12 weeks of subcutaneous implantation, the fibrous capsule of the stent treated with plasma surface modification was formed. Note: (A) The histological section represents the thickness of the fibrous capsule at the interface between the implant and the subcutaneous tissue. Scale bar: 200 μm. (B) The quantification of the thickness of the fibrous capsule showed that the thickness of the fibrous capsule on the unmodified stent was significantly higher than that of the plasma-treated stent (* P <0.05). Abbreviations: Ar, argon; scam, untreated; N2, nitrogen; O2, oxygen.

Figure S8 Schematic diagram of the interaction mechanism between cells and unmodified and plasma surface-modified scaffolds treated with Ar, N2, and O2 plasma. Note: The control scaffold showed lower protein adsorption and cell adhesion, the Ar modified scaffold showed moderate roughness, and the serum protein had higher VN adsorption. Compared with the O2 plasma modified scaffold, the N2 plasma modified scaffold It exhibits higher FN adsorption, with the latter exhibiting the highest surface roughness. The image is out of scale. Abbreviations: Ar, argon; FN, fibronectin; N2, nitrogen; O2, oxygen; VN, vitronectin.

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