Electrospun Patch Functionalized with Nanoparticles Allows for

Subscriber access provided by Gothenburg University Library

Biological and Medical Applications of Materials and Interfaces

Electrospun Patch Functionalized with Nanoparticles Allows for Spatiotemporal Release of VEGF and PDGF-BB Promoting In Vivo Neovascularization Christopher Tsao, Laura Pandolfi, Xin Wang, Silvia Minardi, Cristina Lupo, Michael Evangelopoulos, Troy Hendrickson, Aaron Shi, Gianluca Storci, Francesca Taraballi, and Ennio Tasciotti ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b19975 • Publication Date (Web): 04 Dec 2018 Downloaded from http://pubs.acs.org on December 5, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Electrospun Patch Functionalized with Nanoparticles Allows for Spatiotemporal Release of VEGF and PDGF-BB Promoting In Vivo Neovascularization. Christopher J. Tsaoa, Laura Pandolfia, Xin Wanga, Silvia Minardia, Cristina Lupoa, Michael Evangelopoulosa, Troy Hendricksona,c,Aaron Shia, Gianluca Storcia, Francesca Taraballia,b*and Ennio Tasciottia,b aCenter

for Biomimetic Medicine, Houston Methodist Research Institute, 6670 Bertner Ave.,

Houston, TX 77030, USA bDepartment

of Orthopedics & Sports Medicine, Houston Methodist Hospital, 6550 Fannin St.,

Houston, TX, 77030, USA cMD/PhD

Program, Texas A&M College of Medicine, 8441 Riverside Pkwy., Bryan, TX, 77807

*[email protected], Tel: +1-713-471-9497 (will handle correspondence at all stages) KEYWORDS: Cardiomyocytes, electrospinning, porous silica, neovascularization, growth factors

ACS Paragon Plus Environment

1

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 40

ABSTRACT The use of nanomaterials as carriers for the delivery of growth factors has been applied to a multitude of applications in tissue engineering. However, issues of toxicity, stability and systemic effects of these platforms have yet to be fully understood, especially for cardiovascular applications. Here, we proposed a delivery system composed of poly(DL-lactide-co-glycolide) acid (PLGA) and porous silica nanoparticles (pSi) to deliver vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF). The tight spatiotemporal release of these two proteins has been proven to promote neovascularization.

In order to minimize tissue

toxicity, localize the release, and maintain a stable platform, we conjugated two formulations of PLGA-pSi to electrospun (ES) gelatin to create a combined electrospun patch (ES patch) releasing both PDGF and VEGF. When compared to freely dispersed particles, the ES patch cultured in vitro with neonatal cardiac cells had significantly less particle internalization (2.0±1.3%) compared to free PLGA-pSi (21.5±6.1) or pSi (28.7±2.5) groups. Internalization was positively correlated to late stage apoptosis with PLGA-pSi and pSi groups having increased apoptosis compared to the untreated group. When implanted subcutaneously, the ES patch was shown to have greater neovascularization than controls evidenced by increased expression of α– SMA and CD31 after 21 days. qRT-PCR results support increased angiogenesis by upregulation of VEGFA, VEGFR2, vWF and COL3A1 exhibiting a synergistic effect with the release of VEGF-A164 and PDGF-BB after 21 days in vivo. The results of this study proved that the ES patch reduced cellular toxicity and may be tailored to have a dual release of growth factors promoting localized neovascularization.


2

Page 3 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION In recent years, nanomedicine has attracted much attention in terms of drug delivery applications for tissue engineering. The unique physical and chemical properties of nanoparticles and the various methods used to formulate delivery vehicles can aid in overcoming pharmacological barriers of free drugs or compounds, i.e. bioactivity, solubility, targeting and circulating half-life1-3. However, because of this wide range of nano-based delivery systems, there are no universal assessments of nanotoxicity4. Nano carriers in tissue engineering have numerous applications in delivering biochemical cues that promote implant integration and remodeling of the target tissue. However, cells respond differently to nanoparticles, thus the appropriate delivery platform and application becomes crucial in obtaining the desired physiological effect while minimizing collateral toxicity. In cardiac tissue engineering, the preservation of native cardiac cells is extremely important due to their unique function and terminally differentiated state. Conditions such as a myocardial ischemia and subsequent infarction can lead to necrotic areas of heart tissue due the absence of blood supply. Repair or prevention of infarcted areas with strategies aimed at neovascularization can greatly increase prognosis5-6. Neovascularization can be activated by the spatio-temporal exposure of VEGF and PDGF-BB7-9. It has been shown that release of VEGF is an important regulator for the activation of angiogenesis10 and subsequent PDGF-BB exposure is essential for continued vascular maturation7. Direct injection of growth factors is greatly limited by their short circulation half-life11-12. Therefore, sustained dual delivery of these growth factors is important in tissue engineering applications. The applications of porous silicon or silica have been studied for a number of years and range in numerous fields of study13-15. In terms of drug delivery models, porous silica particles


3


Page 4 of 40

(pSi) have many unique properties such as high loading efficiency, tunability, availability of surface modifications, and ease of synthesis16. Further modifications to pSi release kinetics can be achieved by polymer encapsulations. For years, poly(DL-lactide-co-glycolide) acid (PLGA) has been utilized to encapsulate drugs and other particles because of its biocompatibility and tunable degradation17-18. Delivery of biochemical cues can further be controlled from nano delivery platforms by conjugated particles to a biomaterial substrate19. This ensures localized delivery to targeted area as well as mitigates internalization of free particles. Nanoparticle toxicity is a topic of much debate in recent years. Currently, there are no widely accepted standards for measuring nanoparticle toxicity in cells or in systemic circulation. However, studies have shown negative effects of nanoparticles accumulation20-22. Therefore, the distribution of nanoparticles in the targeted region is crucial to maximize local regenerative potential while minimizing any systemic toxic side effects. This study investigated the angiogenic potential of electrospun (ES) gelatin crosslinked with PLGA-pSi nanoparticles loaded with PDGF-BB and VEGF-A, creating a ES patch. This platform combines materials ranging from nano to millimeters with each component serving a specific purpose. The pSi platform provides the core delivery vehicle which has high loading and growth factor stability. The PLGA coating allows for the tuning and sustained release to promote angiogenesis. Lastly, the ES gelatin core provides for cell attachment and localized growth factor delivery. The pSi are loaded with recombinant mouse VEGF-A isoform 164 and recombinant mouse PDGF-BB. For clarity, VEGF-A isoform 164 for the remainder of the study will be referred to as “VEGF”. Freely dispersed pSi and PLGA-pSi were directly compared to the combined ES patch for internalization and toxicity of neonatal rat cardiac cells in vitro. VEGF and PDGF-BB were both loaded into pSi and coated with different formulations of PLGA in


4

Page 5 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


order to have a sequential and sustained release as previously shown16, 23. Combined ES patches were finally implanted into a subcutaneous BALB/c mouse model and explanted patches were assessed through immunohistology and qRT-PCR.


5


Page 6 of 40

RESULTS ANS DISCUSSION The application of drug delivery in tissue engineering strategies has been widely researched for a number of reparative strategies1,

24-25.

Currently, there are no standardized

regulations for nanoparticle toxicity and assessment due to the myriad of cell types and particle formulations26, but previous studies have shown a strong correlation between cellular toxicity and particle internalization27-29. The limit of diffusion also hinders many tissue engineering strategies leading to implant failure over time caused by inadequate nutrient transfer past 200µm30-31. Ongoing research into prevascularized32-33 and angiogenic34-35 scaffolds have highlighted the need for implantable biomaterials supporting cellular growth and proliferation as well as recruiting native remodeling cascades36. Biomaterials for tissue engineering need to provide both structural support as well as promote native tissue integration. Natural and synthetic scaffolds alone have shown positive results in structural support, but often times do not promote native tissue integration37-38. The key components to tissue integration involve attachment and cellular migration, however these migrated cells will not survive without a linked vasculature network due to limited nutrient diffusion. The promotion of angiogenesis has been linked to a number of signaling molecules and growth factors. However, targeting angiogenesis from an implanted material is challenging without a localized release that can signal vasculogenic cells to the material. Drug releasing materials are a solution to targeting of an implanted biomaterial, but other factors arise such as drug stability, dosage and timing. By encapsulating drugs within delivery vehicles such as nanoparticles, the stability of the drug is preserved and delivery dosage and timing can be tuned based on the encapsulating material16. Additionally, the cellular trafficking of these molecules is crucial to gain the desired cellular response. Growth factors meant to affect an extracellular


6

Page 7 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


receptor will likely have an undesired cellular response if internalized rather than exposed to the surface of the cell39-40. Therefore, internalization of loaded particles is not always a desired effect, particularly in the case of angiogenesis promoted by growth factors. By conjugating drug loaded particles to the surface of the implanted biomaterial, the spatiotemporal delivery kinetics are localized at the scaffold and the surface of cells, which in turn improves the efficacy of this particular delivery system41. In this study, we have shown that a complete ES patch platform, consisting of PLGA-pSi delivering angiogenic growth factors conjugated to the surface of electrospun gelatin, reduced cardiac cell internalization, material toxicity and promoted neovascularization. Ideally, the vehicle for drug or compound delivery in tissue engineering applications would be biocompatible and have a localized payload42. Particle internalization has been shown to have an effect on cellular signaling which in turn can activate apoptotic pathways43. Intracellular trafficking and sorting in different intracellular regions of an individual cell can be dependent on the cell type as well as internalized particle formulation44. The results of this study pertaining to particle internalization of either PLGA-pSi or pSi alone showed to have increased apoptotis. This is likely due to alterations in cellular signaling resulting from the uptake of foreign materials45. However, this increased apoptosis was no present with cells cultured with the combined ES patch The size and characteristics of the particle play an important role in the cellular internalization concentration (Fig 1A, B). For material comparison purposes, ES gelatin alone, without particles, was shown to have fiber diameters in the nanometer range with random fiber orientation (Fig 1C, D). With the addition of particles, SEM images support even PLGA-pSi distribution on the surface of the combined ES patch (ES gelatin with PLGA-pSi particles


7


Page 8 of 40

releasing VEGF and PDGF-BB) and clear distinction of different particle formulations evidenced by their size (Fig 1E). Confocal microscopy demonstrated an even distribution of both formulations of PLGA-pSi conjugated to the surface of the combined ES patches (Fig 1F). Further analysis of confocal images using ImageJ revealed the calculated density of particles conjugated to the surface of ES patches was approximately 402±42 PLGA-pSi-VEGF particles/mm2 and 320±57 PLGA-pSi-PDGF-BB particles/mm2. Averaged particle diameter of 5% 50:50 PLGA-pSi was measured to be 4.3±1.5µm where 10% 75:25 PLGA-pSi produced a larger particle at 8.2±0.8µm (Fig 1G). The sustained and sequential release of VEGF and PDGFBB was tailored based on previously published work7, 9. By choosing a 5% 50:50 PLGA coating for VEGF loaded pSi, the coating was engineered to be thinner and more susceptible to hydrolytic degradation compared to the 10% 75:25 PLGA coating for PDGF-BB loaded pSi. These two polymer coatings were translated in the average particles diameters (Fig 1G) and release profiles (Fig 1J). VEGF was designed to be released first promoting early stages of neovascularization, where the later and sustained release of PDGF-BB was aimed to promote endothelial recruitment and in turn vascular growth and maturation7, 9. The conjugation of PLGA-pSi to ES gelatin did alter the release profiles of PDGF-BB and VEGF in vitro. When coupled with the ES gelatin, each growth factor had a more sustained release over time when compared to release from particles alone (Fig 1J). Due to the engineered particle structures, PLGA-pSi-VEGF particles released nearly 50% payload by day 4 whereas PLGA-pSi-PDGF-BB particles demonstrated a more delayed release with 50% payload delivered by day 8. The conjugation of PLGA-pSi to ES gelatin was not shown to have an impact on the swelling ratio of the material after 7 days (Fig 1H). After loading PDGF-BB and VEGF into their respective PLGA-pSi formulation, the resulting loading efficiency was approximately 70% for both (Fig


8

Page 9 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1I). The loaded PLGA-pSi formulations showed a sustained release of VEGF and PDGF-BB over the course of 21 days. 5% 50:50 PLGA-pSi-VEGF and 10% 75:25 PLGA-pSi-PDGF-BB showed after 15 days to have a cumulative release of 91.2±12.4% and 66.2±1.6%, respectively (Fig 1J). After particles were conjugated to ES gelatin, the cumulative release of VEGF and PDGF-BB was lower than unconjugated particle release after 21 days.


9


Page 10 of 40


10

Page 11 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1: PLGA-pSi and electrospun gelatin characterization. SEM and confocal microscopy of (A,B) PLGA-pSi particles (Red, labeled pSi particles), (scale bar, 10 µm) inset shows pSi alone, (scale bar, 50 µm) (C,D) ES gelatin without particles (scale bar, 100 µm) (E,F) ES patch showing differences in particle size and overall distribution. Both particle formulions were stained with different fluorophores. Green particles represent PLGA-pSi-VEGF particles where violet particles represent PLGA-psi-PDGF-BB particles. (scale bar, 20µm) (G) Average diameter of PLGA-pSi formulations. (H) Swelling ratios of ES patches over time. (I) Loading efficiency of VEGF and PDGF-BB into PLGA-pSi. (J) Release profiles of VEGF and PDGF-BB loaded into PLGA-pSi Looking more closely at the interactions between cardiac cells and the developed materials, it is evident that the particles alone may have been responsible for negative signaling effects. As shown in Figure 2, greater numbers of smaller pSi were able to be internalized by a single cell compared to the larger PLGA-pSi. However, since the goal for this study was to deliver growth factors that affect extracellular signaling, particle internalization was not desirable. Therefore, by conjugating the ES patch, cells were shown to be less able to internalize particles and thus payloads of growth factor were hydrolytically controlled rather than enzymatic digestion from cells. Qualitative analysis of particles internalized by neonatal cardiac cells was visualized by SEM and confocal microscopy. SEM images of PLGA-pSi and pSi groups (Figs 2Aand 2B) showed clear localization of particles on top and below cellular membranes, while cells attached to ES patches revealed minimal particle internalization (Fig 2C). Confocal microscopy supported SEM images showing the localization of freely dispersed FITC labeled PLGA-pSi and pSi particles within cardiac cell cytoskeletons (Figs 2D and 2E). Flow cytometry


11


Page 12 of 40

quantified significantly greater internalization of free PLGA-pSi (21.5±6.1) and pSi (28.7±2.5) compared to the ES patch (2.0±1.3) (Fig 2G).

Figure 2: Neonatal cardiomyocyte particle internalization.

SEM image showing

internalization of (A) pSi, (B) PLGA-pSi and cardiac cells cultured on (C) ES patches. Cardiac cells cultured alone with pSi or PLGA-pSi readily internalized particles into their cytoskeletal structure, whereas cells cultured on ES patches showed little particle internalization. Confocal microscopy images showed corresponding results with (D) pSi and (E) PLGA-pSi particles both co-localized within the cardiac cells. However cardiac cells when cultured on (F) ES patches again showed reduced internalization with little co-localization of particles and cells. (scale bar, 10µm) (G) Results of cardiac cells cultured with pSi, PLGA-psi or ES patch for 24-hours quantifying internalization of particles through flow cytometry. Cells were separated from the particles and patches using the same protocols. Statistical significance was compared between the means of each group through one-way ANOVA followed by Tukey’s test to determine significance (* p < 0.05 is significant, n=3)


12

Page 13 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Apoptosis and necrosis quantified through flow cytometry revealed no significance between groups tested on day 0 with 30 min exposure to particles (Fig 3A). This may have resulted from inherent apoptotic signaling due to incomplete cellular attachment. It should be noted that cell retrieval from the combined ES Patch (ES gelatin with PLGA-pSi releasing VEGF and PDGF-BB) was a stringent procedure (see methods for detailed protocol), but this procedure was performed at each time point for every experimental and control group. The exposure to collagenase, the chelating agent and agitation during cell isolation may have also played an inherent role in the overall apoptotic state. Days 1 and 3 revealed significant differences in total apoptosis (Fig 3B) when compared to untreated control. At day 3 only the ES patch group was shown to have a significant decrease in the total necrosis percentage (Fig 3C). However when the total apoptosis/necrosis percentages are analyzed, the double positive cell population of Annexin V and propidium iodide, day 1 revealed that there were significantly higher late stage apoptosis in both free particle groups compared to untreated (Fig 3D). Where PLGA-pSi and pSi groups showed 17.6±6.6% and 14.7±4.2%, respectively, ES patches had only 3.0±1.2% late stage apoptosis. After 3 days of particle exposure, both PLGA-pSi and pSi had internalization comparable to the control, but the combined ES patch was significantly lower (4.1±0.5%). This may have influenced the total apoptosis percentage in that a significant population of cells have already lysed and were fragmented. However, the ES patch proved to decrease total apoptosis and have the highest percentage of viable cells.


13


Page 14 of 40

Figure 3: Apoptosis resulting from particle exposure. Flow cytometry analyzed different stages of apoptosis/necrosis in neonatal cardiac cells based on specific cell-material interactions. Untreated group represents cardiac cells not exposed to particles or combined ES patch. Each group was isolated using the same cell dissociation protocols procedures. (A) Annexin V and propidium iodide results of cardiac cells exposed to PLGA-pSi and pSi particles alone as well as the combined ES patch at days 0, 1 and 3. All groups except the ES Patch were


14

Page 15 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


shown to have an increased populations of apoptosis/necrosis over the 3 day period. (B) Total apoptosis, (C) total necrosis and (D) double positive apoptosis & necrosis percentages over time compared to untreated control group. Statistical significance was compared between the means of each group to the untreated group within a given time point through two-way ANOVA followed by Tukey’s multiple comparison post hoc test. (* p < 0.05 is significant, n=3) Gross observation of combined ES patch explants showed visually significant differences compared to patches with either growth factor alone, suggesting a synergistic vascularization effect. After 7 days in vivo, no group had observable vascularization (Fig 4A, 4B and 4C). Observable red areas surrounding the patch area were only detectable by gross analysis in the combined ES patch after 14 days (Fig 4F and 4I), suggesting the possibility of angiogenesis which could be confirmed by molecular analysis. Immunohistological analysis of explanted combined ES patches showed significantly higher expression of neovasculogenic markers, αSMA and CD31, after 21 days in vivo (Fig 5). Blank controls, ES gelatin with PLGA-pSi particles without growth factors, revealed slight α–SMA expression within the patch after 21 days (Fig 5B). In the ES patch there was greater α–SMA and CD31 expression at 7, 14, and 21 days compared to the blank control (Figs 5B, C). Measured immunofluorescent expression of CD31 was significant for all time points in the dual release group compared to the control at all time points. At 14 and 21 days of the growth factor releasing patches, the co-localization of α– SMA and CD31 was observed, signaling neovascularization. Higher magnification histology images confirm the increased α–SMA and CD31 expression in the combined ES patch after 21 days suggesting increased angiogenesis. Relative lumen quantification revealed nearly a two-fold increase in the colocalization of α-SMA and CD31 at all timepoints (Fig 5D), signifying increased angiogenic activity compared to the negative control and either growth factor alone.


15


Page 16 of 40

Lumen formations are larger and more frequent in the 21-day time points compared to 14 days suggesting that the timing of growth factor release was sufficient to promote sustained vessel maturation. The sustained release proved to be beneficial in the neovascularization process in vivo due to continued vessel maturation in tissue remodeling. The localization of particles to the patch area also minimized nonspecific distribution of growth factors limiting any significant systemic effects in vivo.

Figure 4: Gross observations of subcutaneous patch explants up to 21 days in vivo. Comparison of patches containing each of the growth factors alone compared to the ES patch containing both PDGF-BB and VEGF tuned to have a sequential release alludes to a temporal synergistic effect in terms of neovascularization. Patches containing conjugated PLGA-pSiPDGF-BB alone (A, D and G), VEGF-PLGA-pSi alone (B, E and H) and the ES patch (C, F, I)


16

Page 17 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


were excised from mice 7, 14 and 21 days. Patches were observed visually for increased vascularization near the site of implant.

Figure 5: In vivo immunohistological sections of implanted ES patches up to 21 days. (A) Sections were stained for α-SMA (green) and CD31 (red). Dotted lines represent border of combined ES patches. Dark areas represent locations of previously explanted patches. Bottom row of histology panel shows increased magnification of tissues adjacent to implanted patch area (scale bar, 20µm) (B, C) Quantification of α-SMA and CD31 (D) Lumen quantification analysis normalized to control. Statistical significance was compared between the means of each group to the blank within a given time point through two-way ANOVA followed by Tukey’s multiple comparison post hoc test. (* p < 0.05, ** p

Electrospun Patch Functionalized with Nanoparticles Allows for

Recommend Documents