Electrospun electroconductive constructs of aligned fibers for cardiac tissue engineering.

Myocardial infarction remains the leading cause of death in the western world. Since the heart has limited regenerative capabilities, several cardiac tissue engineering (CTE) strategies have been proposed to repair the damaged myocardium. A novel electrospun construct with aligned and electroconductive fibers combining gelatin, poly(lactic-co-glycolic) acid and polypyrrole that may serve as a cardiac patch is presented. Constructs were characterized for fiber alignment, surface wettability, shrinkage and swelling behavior, porosity, degradation rate, mechanical properties, and electrical properties. Cell-biomaterial interactions were studied using three different types of cells, Neonatal Rat Ventricular Myocytes (NRVM), human lung fibroblasts (MRC-5) and induced pluripotent stem cells (iPSCs). All cell types showed good viability and unique organization on construct surfaces depending on their phenotype. Finally, we assessed the maturation status of NRVMs after 14 days by confocal images and qRT-PCR. Overall evidence supports a proof-of-concept that this novel biomaterial construct could be a good candidate patch for CTE applications.

Endosomal Escape of Polymer-Coated Silica Nanoparticles in Endothelial Cells.

Current investigations into hazardous nanoparticles (i.e., nanotoxicology) aim to understand the working mechanisms that drive toxicity. This understanding has been used to predict the biological impact of the nanocarriers as a function of their synthesis, material composition, and physicochemical characteristics. It is particularly critical to characterize the events that immediately follow cell stress resulting from nanoparticle internalization. While reactive oxygen species and activation of autophagy are universally recognized as mechanisms of nanotoxicity, the progression of these phenomena during cell recovery has yet to be comprehensively evaluated. Herein, primary human endothelial cells are exposed to controlled concentrations of polymer-functionalized silica nanoparticles to induce lysosomal damage and achieve cytosolic delivery. In this model, the recovery of cell functions lost following endosomal escape is primarily represented by changes in cell distribution and the subsequent partitioning of particles into dividing cells. Furthermore, multilamellar bodies are found to accumulate around the particles, demonstrating progressive endosomal escape. This work provides a set of biological parameters that can be used to assess cell stress related to nanoparticle exposure and the subsequent recovery of cell processes as a function of endosomal escape.

Cross-linking of porcine acellular dermal matrices negatively affects induced neovessel formation using platelet-rich plasma in a rat model of hernia repair.

The degree of cross-linking within acellular dermal matrices (ADM) seems to correlate to neovascularization when used in ventral hernia repair (VHR). Platelet-rich plasma (PRP) enhances wound healing through several mechanisms including neovascularization, but research regarding its effect on soft tissue healing in VHR is lacking. We sought to study the effect of cross-linking on PRP-induced neovascularization in a rodent model of bridging VHR. We hypothesized that ADM cross-linking would negatively affect PRP-induced neovessel formation. PRP was extracted and characterized from pooled whole blood. Porcine cross-linked (cADM) and non-cross-linked ADMs (ncADM) were implanted in a rat model of chronic VHR after treatment with saline (control) or PRP. Neovascularization of samples at 2, 4, and 6 weeks was assessed by hematoxylin and eosin and immunohistochemical staining of CD 31. Adhesion severity at necropsy was compared using a previously validated scale. Addition of PRP increased neovascularization in both cADM and ncADM at 2- and 4-week time points but appeared to do so in a dependent fashion, with significantly greater neovascularization in the PRP-treated ncADMs compared to cADMs. Omental adhesions were increased in all PRP-treated groups. Results indicate that, for 2-week measurements when compared with the cADM group without PRP therapy, the mean change in neovascularization due to ncADM was 3.27 (Z = 2.75, p = 0.006), PRP was 17.56 (Z = 14.77, p < 0.001), and the combined effect of ncADM and PRP was 9.41 (Z = 5.6, p < 0.001). The 4-week data indicate that the average neovascularization change due to ncADM was 0.676 (Z = 0.7, p = 0.484), PRP was 7.69 (Z = 7.95, p < 0.001), and combined effect of ncADM and PRP was 5.28 (Z = 3.86, p < 0.001). These findings validate PRP as a clinical adjunct to enhance the native tissue response to implantable biomaterials and suggest that ncADM is more amenable than cADM to induced neovascularization. PRP use could be advantageous in patients undergoing VHR where poor incorporation is anticipated and early-enhanced neovascularization is desired.

Increased use of surgical energy promotes methicillin-resistant Staphylococcus aureus colonization in rabbits following open ventral hernia mesh repair.

BACKGROUND: Surgical energy has been widely implemented because of ease of use, effective hemostasis, and surgical dissection. Studies demonstrate its use to be an independent risk factor for postoperative wound infection. Methicillin-resistant Staphylococcus aureus (MRSA) is the most common bacteria found in postoperative mesh infection. No reports are available on the sequelae of surgical energy use for open ventral hernia repair (oVHR) with mesh. We hypothesized that increasing amounts of surgical energy will result in higher infectious burden after oVHR with composite multifilament polyester mesh (Parietex™ PCO). METHODS: New Zealand rabbits underwent bridging oVHR with Parietex™ PCO and were divided into three surgical treatment groups: (1) scalpel alone, (2) 120 J of energy, and (3) 600 J of energy. The bioprosthesis was then inoculated with 105 colony-forming units of MRSA. Rabbits were survived for 7 days with daily physical examination. Complete blood count, basci metabolic panel, and blood cultures were performed on postoperative days one, four, and seven. Surviving rabbits were killed, and meshes explanted for MRSA colony counts. RESULTS: Rabbits receiving the most surgical energy developed signs and symptoms of severe sepsis and wound necrosis within 24 h. In comparison, rabbits receiving no surgical energy had significantly less MRSA recovered from explanted mesh, significantly less bacteremia, and fewer adhesions. CONCLUSIONS: Increased use of surgical energy promoted greater colonization, exaggerated septic response to bacterial contamination, and more severe adhesions. In the absence of devitalized tissue, rabbits can effectively limit bacterial contamination. These findings support the surgical principles of proper tissue handling and highlight the detrimental effects of indiscriminant surgical energy usage, thus emphasizing the importance of programs such as Fundamental Use of Surgical Energy.

Nanocomposite Hydrogels as Platform for Cells Growth, Proliferation, and Chemotaxis.

The challenge of mimicking the extracellular matrix with artificial scaffolds that are able to reduce immunoresponse is still unmet. Recent findings have shown that mesenchymal stem cells (MSC) infiltrating into the implanted scaffold have effects on the implant integration by improving the healing process. Toward this aim, a novel polyamidoamine-based nanocomposite hydrogel is synthesized, cross-linked with porous nanomaterials (i.e., mesoporous silica nanoparticles), able to release chemokine proteins. A comprehensive viscoelasticity study confirms that the hydrogel provides optimal structural support for MSC infiltration and proliferation. The efficiency of this hydrogel, containing the chemoattractant stromal cell-derived factor 1α (SDF-1α), in promoting MSC migration in vitro is demonstrated. Finally, subcutaneous implantation of SDF-1α-releasing hydrogels in mice results in a modulation of the inflammatory reaction. Overall, the proposed SDF-1α-nanocomposite hydrogel proves to have potential for applications in tissue engineering.

Biomimetic Concealing of PLGA Microspheres in a 3D Scaffold to Prevent Macrophage Uptake.

Scaffolds functionalized with delivery systems for the release of growth factors is a robust strategy to enhance tissue regeneration. However, after implantation, macrophages infiltrate the scaffold, eventually initiating the degradation and clearance of the delivery systems. Herein, it is hypothesized that fully embedding the poly(d,l-lactide-co-glycolide acid) microspheres (MS) in a highly structured collagen-based scaffold (concealing) can prevent their detection, preserving the integrity of the payload. Confocal laser microscopy reveals that non-embedded MS are easily internalized; when concealed, J774 and bone marrow-derived macrophages (BMDM) cannot detect them. This is further demonstrated by flow cytometry, as a tenfold decrease is found in the number of MS engulfed by the cells, suggesting that collagen can cloak the MS. This correlates with the amount of nitric oxide and tumor necrosis factor-α produced by J774 and BMDM in response to the concealed MS, comparable to that found for non-functionalized collagen scaffolds. Finally, the release kinetics of a reporter protein is preserved in the presence of macrophages, only when MS are concealed. The data provide detailed strategies for fabricating three dimensional (3D) biomimetic scaffolds able to conceal delivery systems and preserve the therapeutic molecules for release.

Infusion of iodine-based contrast agents into poly(p-dioxanone) as a radiopaque resorbable IVC filter.

To determine the feasibility of infusing resorbable inferior vena cava (IVC) filter with iodine-based contrast agents to produce a radiopaque, computed tomography (CT)-visible IVC filter. Infused poly(p-dioxanone) (PPDO) was obtained by incubating PPDO in different concentrations of 4-iodobenzoyl chloride (IBC) and 2,3,5-triiodobenzoic acid (TIBA). Characterizations of infused and nascent PPDO were done using elemental analysis, micro-CT, tensile strength analysis, scanning electron microscopy, and differential scanning calorimetry. Elemental analysis showed percentage loading of 1.07 ± 0.08 for IBC and 0.73 ± 0.01 for TIBA. The iodine loading remained the same within 2 weeks for TIBA but decreased to about 80 % with IBC when subjected to physiological conditions. Micro-CT images showed increased attenuation of the infused PPDO compared with the nascent PPDO. The Hounsfield unit values for infused and nascent sutures were 110 ± 40 and 153 ± 53 for PPDO infused with 2 mg/mL IBC and TIBA, respectively, but only 11.35 ± 2 for nascent PPDO. In contrast the HU for bone was 116 ± 37. Tensile strength analysis showed maximum loads of 1.01 ± 0.43 kg and 10.02 ± 0.54 kg for IBC and TIBA, respectively, and 10.10 ± 0.64 kg for nascent PPDO. Scanning electron microscopy showed that the morphology of the PPDO surface did not change after coating and preliminary cytotoxicity assay showed no killing effect on Hela cells. PPDO infused with a contrast agent is significantly more radiopaque than nascent PPDO on micro-CT imaging. This radiopacity could allow the position and integrity of infused resorbable IVC filter to be monitored while it is in place, thus increasing its safety and efficacy as a medical device.

Multiscale patterning of a biomimetic scaffold integrated with composite microspheres.

The ideal scaffold for regenerative medicine should concurrently mimic the structure of the original tissue from the nano- up to the macroscale and recapitulate the biochemical composition of the extracellular matrix (ECM) in space and time. In this study, a multiscale approach is followed to selectively integrate different types of nanostructured composite microspheres loaded with reporter proteins, in a multi-compartment collagen scaffold. Through the preservation of the structural cues of the functionalized collagen scaffold at the nano- and microscale, its macroscopic features (pore size, porosity, and swelling) are not altered. Additionally, the spatial confinement of the microspheres allows the release of the reporter proteins in each of the layers of the scaffold. Finally, the staged and zero-order release kinetics enables the temporal biochemical patterning of the scaffold. The versatile manufacturing of each component of the scaffold results in the ability to customize it to better mimic the architecture and composition of the tissues and biological systems.

Nanotechnologies and regenerative medical approaches for space and terrestrial medicine.

One purpose of the International Space Station (ISS) is to explore powerful new areas of biomedical science in microgravity. Recent advances in nanotechnology applied to medicine--what we now refer to as nano-medicine--and regenerative medicine have enormous untapped potential for future space and terrestrial medical applications. Novel means for drug delivery and nanoscale screening tools will one day benefit astronauts venturing to Mars and places beyond, while the space laboratory will foster advances in nanotechnologies for diagnostic and therapeutic tools to help our patients here on Earth. Herein we review a series of nanotechnologies and selected regenerative medical approaches and highlight key areas of ongoing and future investigation that will benefit both space and terrestrial medicine. These studies target significant areas of human disease such as osteoporosis, diabetes, radiation injury, and many others.

Agarose surface coating influences intracellular accumulation and enhances payload stability of a nano-delivery system.

PURPOSE: Protein therapeutics often require repeated administrations of drug over a long period of time. Protein instability is a major obstacle to the development of systems for their controlled and sustained release. We describe a surface modification of nanoporous silicon particles (NSP) with an agarose hydrogel matrix that enhances their ability to load and release proteins, influencing intracellular delivery and preserving molecular stability. METHODS: We developed and characterized an agarose surface modification of NSP. Stability of the released protein after enzymatic treatment of loaded particles was evaluated with SDS-page and HPLC analysis. FITC-conjugated BSA was chosen as probe protein and intracellular delivery evaluated by fluorescence microscopy. RESULTS: We showed that agarose coating does not affect NSP protein release rate, while fewer digestion products were found in the released solution after all the enzymatic treatments. Confocal images show that the hydrogel coating improves intracellular delivery, specifically within the nucleus, without affecting the internalization process. CONCLUSIONS: This modification of porous silicon adds to its tunability, biocompatibility, and biodegradability the ability to preserve protein integrity during delivery without affecting release rates and internalization dynamics. Moreover, it may allow the silicon particles to function as protein carriers that enable control of cell function.

Addition of platelet-rich plasma supports immune modulation and improved mechanical integrity in Alloderm mesh for ventral hernia repair in a rat model.

The recurrence of ventral hernias continues to be a problem faced by surgeons, in spite of efforts toward implementing novel repair techniques and utilizing different materials to promote healing. Cadaveric acellular dermal matrices (Alloderm) have shown some promise in numerous surgical subspecialties, but these meshes still suffer from subsequent failure and necessitation of re-intervention. Here, it is demonstrated that the addition of platelet rich plasma to Alloderm meshes temporally modulates both the innate and cytotoxic inflammatory responses to the implanted material. This results in decreased inflammatory cytokine production at early time points, decreased matrix metalloproteinase expression, and decreased CD8+ T cell infiltration. Collectively, these immune effects result in a healing phenotype that is free from mesh thinning and characterized by increased material stiffness.

Polyester Mesh Functionalization with Nitric Oxide-Releasing Silica Nanoparticles Reduces Early Methicillin-Resistant Staphylococcus aureus Contamination.

Background: Infected hernia mesh is a cause of post-operative morbidity. Nitric oxide (NO) plays a key role in the endogenous immune response to infection. We sought to study the efficacy of a NO-releasing mesh against methicillin-resistant Staphylococcus aureus (MRSA). We hypothesized that a NO-releasing polyester mesh would decrease MRSA colonization and proliferation. Materials and Methods: A composite polyester mesh functionalized with N-diazeniumdiolate silica nanoparticles was synthesized and characterized. N-diazeniumdiolate silica parietex composite (NOSi) was inoculated with 104,106, or 108 colony forming units (CFUs) of MRSA and a dose response was quantified in a soy tryptic broth assay. Utilizing a rat model of contaminated hernia repair, implanted mesh was inoculated with MRSA, recovered, and CFUs were quantified. Clinical metrics of erythema, mesh contracture, and adhesion severity were then characterized. Results: Methicillin-resistant Staphylococcus aureus CFUs demonstrated a dose-dependent response to NOSi in vitro. In vivo, quantified CFUs showed a dose-dependent response to NOSi-PCO. Treated rats had fewer severe adhesions, less erythema, and reduced mesh contracture. Conclusions: We demonstrate the efficacy of a NO-releasing mesh to treat MRSA in vitro and in vivo. Creation of a novel class of antimicrobial prosthetics offers new strategies for reconstructing contaminated abdominal wall defects and other procedures that benefit from deploying synthetic prostheses in contaminated environments.

Lysyl oxidase engineered lipid nanovesicles for the treatment of triple negative breast cancer.

In the field of oncology research, a deeper understanding of tumor biology has shed light on the role of environmental conditions surrounding cancer cells. In this regard, targeting the tumor microenvironment has recently emerged as a new way to access this disease. In this work, a novel extracellular matrix (ECM)-targeting nanotherapeutic was engineered using a lipid-based nanoparticle chemically linked to an inhibitor of the ECM-related enzyme, lysyl oxidase 1 (LOX), that inhibits the crosslinking of elastin and collagen fibers. We demonstrated that, when the conjugated vesicles were loaded with the chemotherapeutic epirubicin, superior inhibition of triple negative breast cancer (TNBC) cell growth was observed both in vitro and in vivo. Moreover, in vivo results displayed prolonged survival, minimal cytotoxicity, and enhanced biocompatibility compared to free epirubicin and epirubicin-loaded nanoparticles. This all-in-one nano-based ECM-targeting chemotherapeutic may provide a key-enabling technology for the treatment of TNBC.

Electrospun anti-inflammatory patch loaded with essential oils for wound healing.

Wound healing is a complex process that consists of three overlapping phases: inflammation, proliferation, and remodeling. A bacterial infection can increase inflammation and delay this process. Microorganisms are closely related to the innate immune system, such as macrophages and neutrophils, as they can start an inflammatory cascade. Essential oils play an important role in the inhibition and prevention of bacterial growth due to their ability to reduce antimicrobial resistance. The possibility to find a strategy that combines antimicrobial and anti-inflammatory properties is particularly appealing for wound healing. In this work, we showcase a variety of patches based on electrospun polycaprolactone (PCL) nanofibers loaded with natural compounds derived from essential oils, such as thymol (THY) and tyrosol (TYR), to achieve reduced inflammation. In addition, we compared the effect these essential oils have on activated macrophages when incorporated into the PCL patch. Specifically, we demonstrate that PCL-THY resulted in more efficient down-regulation of pro-inflammatory genes related to the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κb) pathway when compared to PCL-TYR and the combination patch containing TYR and THY (i.e., PCL-TYR-THY). Furthermore, PCL-THY displayed low affinity for cell attachment, which may hinder wound adherence and integration. Overall, our results indicate that THY-loaded patches could serve as promising candidates for the fabrication of dressings that incorporate bactericidal and anti-inflammatory properties while simultaneously avoiding the limitations of traditional antibiotic-loaded devices.

Leukocyte-mimicking nanovesicles for effective doxorubicin delivery to treat breast cancer and melanoma.

In the last decades, several approaches were developed to design drug delivery systems to address the multiple biological barriers encountered after administration while safely delivering a payload. In this scenario, bio-inspired and bio-mimetic approaches have emerged as promising solutions to evade the mononuclear phagocytic system while simultaneously negotiating the sequential transport across the various biological barriers. Leukocytes freely circulate in the bloodstream and selectively target the inflamed vasculature in response to injury, infection, and cancer. Recently we have shown the use of biomimetic nanovesicles, called leukosomes, which combine both the physical and biological properties of liposomes and leukocytes, respectively, to selectively deliver drugs to the inflamed vasculature. Here we report the use of leukosomes to target and deliver doxorubicin, a model chemotherapeutic, to tumors in syngeneic murine models of breast cancer and melanoma. Exploiting the inflammatory pathway responsible for recruiting immune cells to the site of injury, leukosomes exhibited increased targeting of cancer vasculature and stroma. Furthermore, delivery of doxorubicin with leukosomes enabled significant tumor growth inhibition compared with free doxorubicin in both breast and melanoma tumors. This study demonstrates the promise of using biomimetic nanovesicles for effective cancer management in solid tumors.

Biomimetic cellular vectors for enhancing drug delivery to the lungs.

Despite recent advances in drug delivery, the targeted treatment of unhealthy cells or tissues continues to remain a priority. In cancer (much like other pathologies), delivery vectors are designed to exploit physical and biological features of unhealthy tissues that are not always homogenous across the disease. In some cases, shifting the target from unhealthy tissues to the whole organ can represent an advantage. Specifically, the natural organ-specific retention of nanotherapeutics following intravenous administration as seen in the lung, liver, and spleen can be strategically exploited to enhance drug delivery. Herein, we outline the development of a cell-based delivery system using macrophages as a delivery vehicle. When loaded with a chemotherapeutic payload (i.e., doxorubicin), these cellular vectors (CELVEC) were shown to provide continued release within the lung. This study provides proof-of-concept evidence of an alternative class of biomimetic delivery vectors that capitalize on cell size to provide therapeutic advantages for pulmonary treatments.

Microfluidic Assembly of Liposomes with Tunable Size and Coloading Capabilities.

Liposomes used for the delivery of pharmaceuticals have difficulties scaling up and reaching clinical translation as they suffer from batch-to-batch variability. Here, we describe a microfluidic approach for creating reproducible, homogenous nanoparticles with tunable characteristics. These nanoparticles of sizes ranging from 30 to 500 nm are rapidly self-assembled by controlling the flow rates of ethanol and aqueous streams. This method of microfluidic assembly allows for the efficient encapsulation of both hydrophobic and hydrophilic drugs in the lipid bilayer and particle core, respectively, either separately or in combination.

Biomimetic Tissue Engineering: Tuning the Immune and Inflammatory Response to Implantable Biomaterials.

Regenerative medicine technologies rely heavily on the use of well-designed biomaterials for therapeutic applications. The success of implantable biomaterials hinges upon the ability of the chosen biomaterial to negotiate with the biological barriers in vivo. The most significant of these barriers is the immune system, which is composed of a highly coordinated organization of cells that induce an inflammatory response to the implanted biomaterial. Biomimetic platforms have emerged as novel strategies that aim to use the principle of biomimicry as a means of immunomodulation. This principle has manifested itself in the form of biomimetic scaffolds that imitate the composition and structure of biological cells and tissues. Recent work in this area has demonstrated the promising potential these technologies hold in overcoming the barrier of the immune system and, thereby, improve their overall therapeutic efficacy. In this review, a broad overview of the use of these strategies across several diseases and future avenues of research utilizing these platforms is provided.

Controlled Release of Small Molecules for Cardiac Differentiation of Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) have been shown to differentiate to functional cardiomyocytes (CM) with high efficiency through temporally controlled inhibition of the GSK3/Wnt signaling pathways. In this study, we investigated the ability of temporally controlled release of GSK3/Wnt small-molecule inhibitors to drive cardiac differentiation of iPSC without manual intervention. Porous silica particles were loaded with GSK3 inhibitor CHIR99021 or Wnt inhibitor IWP2, and the particles containing IWP2 were coated with 5 wt% poly(lactic-co-glycolic acid) 50:50 to delay release by ∼72 h. iPSCs reprogrammed through mRNA transfection were cultured with these particles up to 30 days. High-performance liquid chromatography suggests a burst release of CHIR99021 within the first 24 h and a delayed release of IWP2 after 72 h. Annexin V/propidium iodide staining did not show a significant effect on apoptosis or necrosis rates. Cultured cells upregulated both early (Nkx 2.5, Isl-1) and late (cTnT, MHC, Cx43) cardiac markers, assayed with a quantitative real-time polymerase chain reaction, and began spontaneous contraction at 3.0 ± 0.6 Hz at 15-21 days after the start of differentiation. CM had clear sarcomeric striations when stained for ß-myosin heavy chain, and showed expression and punctate membrane localization of gap junction protein Connexin43. Calcium and voltage-sensitive imaging showed both action potential and calcium transients typical of immature CM. This study showed that the cardiac differentiation of pluripotent stem cells can be directed by porous silica vectors with temporally controlled release of small-molecule inhibitors. These results suggest methods for automating and eliminating variability in manual maintenance of inhibitor concentrations in the differentiation of pluripotent stem cells to CM.

Electrospun Patch Functionalized with Nanoparticles Allows for Spatiotemporal Release of VEGF and PDGF-BB Promoting In Vivo Neovascularization.

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 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. Quantitative reverse transcription-polymerase chain reaction results support increased angiogenesis by the 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.