An electrospun macrodevice for durable encapsulation of human cells with consistent secretion of therapeutic antibodies.

Frequent subcutaneous or intravenous administrations of therapeutic biomolecules can be costly and inconvenient for patients. Implantation of encapsulated recombinant cells represents a promising approach for the sustained delivery of biotherapeutics. However, foreign body and fibrotic response against encapsulation materials results in drastically reduced viability of encapsulated cells, presenting a major engineering challenge for biocompatibility. Here, we show that the multi-laminate electrospun retrievable macrodevice (Bio-Spun) protects genetically modified human cells after subcutaneous implant in mice. We describe here a biocompatible nanofiber device that limits fibrosis and extends implant survival. For more than 150 days, these devices supported human cells engineered to secrete the antibodies: vedolizumab, ustekinumab, and adalimumab, while eliciting minimal fibrotic response in mice. The porous electrospun cell chamber allowed secretion of the recombinant antibodies into the host bloodstream, and prevented infiltration of host cells into the chamber. High plasma levels (>50 µg/mL) of antibody were maintained in the optimized devices for more than 5 months. Our findings demonstrate that macrodevices constructed from electrospun materials are effective in protecting genetically engineered cells for the sustained administration of recombinant therapeutic antibodies.

Cytotoxicity associated with electrospun polyvinyl alcohol.

Polyvinyl alcohol (PVA) is a synthetic, water-soluble polymer, with applications in industries ranging from textiles to biomedical devices. Research on electrospinning of PVA has been targeted toward optimizing or finding novel applications in the biomedical field. However, the effects of electrospinning on PVA biocompatibility have not been thoroughly evaluated. In this study, the cytotoxicity of electrospun PVA (nPVA) which was not crosslinked after electrospinning was assessed. PVA polymers of several molecular weights were dissolved in distilled water and electrospun using the same parameters. Electrospun PVA materials with varying molecular weights were then dissolved in tissue culture medium and directly compared against solutions of nonelectrospun PVA polymer in human coronary artery smooth muscle cells and human coronary artery endothelial cells cultures. All nPVA solutions were cytotoxic at a threshold molar concentration that correlated with the molecular weight of the starting PVA polymer. In contrast, none of the nonelectrospun PVA solutions caused any cytotoxicity, regardless of their concentration in the cell culture. Evaluation of the nPVA material by differential scanning calorimetry confirmed that polymer degradation had occurred after electrospinning. To elucidate the identity of the nPVA component that caused cytotoxicity, nPVA materials were dissolved, fractionated using size exclusion columns, and the different fractions were added to HCASMC and human coronary artery endothelial cells cultures. These studies indicated that the cytotoxic component of the different nPVA solutions were present in the low-molecular-weight fraction. Additionally, the amount of PVA present in the 3-10 kg/mol fraction was approximately sixfold greater than that in the nonelectrospun samples. In conclusion, electrospinning of PVA resulted in small-molecular-weight fractions that were cytotoxic to cells. This result demonstrates that biocompatibility of electrospun biodegradable polymers should not be assumed on the basis of success of their nonelectrospun predecessors.

Gene silencing in human aortic smooth muscle cells induced by PEI-siRNA complexes released from dip-coated electrospun poly(ethylene terephthalate) grafts.

An excessive tissue response to prosthetic arterial graft material leads to intimal hyperplasia (IH), the leading cause of late graft failure. Seroma and abnormal capsule formation may also occur after prosthetic material implantation. The matricellular protein Thrombospondin-2 (TSP-2) has shown to be upregulated in response to biomaterial implantation. This study evaluates the uptake and release of small interfering RNA (siRNA) from unmodified and surface functionalized electrospun PET graft materials. ePET graft materials were synthesized using electrospinning technology. Subsets of the ePET materials were then chemically modified to create surface functional groups. Unmodified and surface-modified ePET grafts were dip-coated in siRNAs alone or siRNAs complexed with transfection reagents polyethyleneimine (PEI) or Lipofectamine RNAiMax. Further, control and TSP-2 siRNA-PEI complex treated ePET samples were placed onto a confluent layer of human aortic smooth muscle cells (AoSMCs). Complexation of all siRNAs with PEI led to a significant increase in adsorption to unmodified ePET. TSP-2 siRNA-PEI released from unmodified-ePET silenced TSP-2 in AoSMC. Regardless of the siRNA-PEI complex evaluated, AoSMC migrated into the ePET. siRNA-PEI complexes delivered to AoSMC from dip-coated ePET can result in gene knockdown. This methodology for siRNA delivery may improve the tissue response to vascular and other prosthetics.

Fibronectin adsorption on functionalized electrospun polycaprolactone scaffolds: experimental and molecular dynamics studies.

Designing scaffolds to modulate protein adsorption is a key to building advanced scaffolds for tissue regeneration. Protein adsorption to tissue engineering scaffolds is critical in early cell attachment, survival, and eventual proliferation. The goal of this study is to examine the effect of functionalization on fibronectin adsorption to electrospun polycaprolactone (PCL) scaffolds through experimentation using fluorescently labeled fibronectin and to couple this experimental data with analysis of interaction energies obtained through molecular dynamics (MD) simulations to develop a better understanding of the adsorption process. This study is the first to analyze and compare experimental and MD simulation results of fibronectin adsorption on functionalized electrospun PCL scaffolds. Electrospun nanofiber PCL scaffolds were treated with either 1 N NaOH (hydrolyzed) or 46% hexamethylenediamine (HMD) (aminated) solution to be compared with untreated (control) scaffolds. We found that aminated PCL scaffolds experimentally adsorbed more fibronectin than control scaffolds, whereas hydrolyzed scaffolds showed decreased adsorption. MD simulations carried out with NVT ensemble at a temperature of 310 K indicated a higher work of adhesion for both functionalized scaffolds over control. Also, the simulations revealed different conformations of fibronectin on each scaffold type after adsorption, with the arginine-glycine-aspartic acid sequence appearing most accessible on the aminated scaffolds. This suggests that functionalization affects not only the quantity of protein that will adsorb on a scaffold but how it attaches as well, which could affect subsequent cell attachment.

Use of textile dyeing technology to create an infection-resistant functionalized polyester biomaterial.

Infection is a major complication when utilizing implantable devices. The purpose of this study was to create a functionalized polyethylene terephthalate (polyester) biomaterial with sustained antimicrobial properties using textile-dyeing technology. Polyester was hydrolyzed via exposure to sodium hydroxide (NaOH) to provide two functional sites within the polymeric backbone. A modified textile dyeing technique known as thermofixation or pad-heating (pad-heat) in conjunction with autoclaving was employed to directly incorporate the fluoroquinolone antibiotic Ciprofloxacin (Cipro) into polyester fibers. Woven polyester segments were placed into various concentrations of boiling NaOH solutions to create carboxylic acid and hydroxyl groups (HYD). The segments were then sprayed (padded) with a 5 mg mL(-1) Cipro solution and dried overnight, followed by exposure to intense heat and autoclaving. Untreated HYD, Cipro-dipped, and pad-heat-treated HYD segments were then washed under stringent conditions. The antimicrobial activity of the each material was determined via zone of inhibition. Untreated HYD controls had no antimicrobial activity at any of the time periods examined. Cipro-dipped HYD segments had no antimicrobial activity after 1 h. In contrast, antimicrobial activity for autoclaved, pad-heat-treated HYD segments persisted for 80 days (length of study). Autoclave usage prior to plating affected antimicrobial activity substantially. Additionally, varying hydrolysis concentrations did not significantly affect overall Cipro release. Thus, Cipro application to HYD polyester via thermofixation resulted in controlled, sustained antibiotic release over an extended period of time. The long-term infection resistance provided by this technique may address major problems of infection from which implantable devices suffer.

In vivo testing of an infection-resistant annuloplasty ring.

BACKGROUND: An infection-resistant surface incorporated into a prosthetic cardiac valve has great potential clinical applications. STUDY DESIGN: A sewing ring construct was created using ciprofloxacin-treated polyester. Then ciprofloxacin-treated and untreated constructs were implanted subcutaneously on the dorsum of rats and inoculated with Staphylococcus aureus. At 7, 14, and 30 days animals were sacrificed and the implants were retrieved. Each implant was assessed for frank purulence and gross tissue incorporation by a blinded observer. The implants were processed for conventional histology and examined by a blinded Pathologist. Ciprofloxacin-treated rings were also implanted in the absence of a bacterial challenge. At explantation, a maximal zone of inhibition, if present, was measured. Finally, ciprofloxacin was eluted with methanol from the explanted segments and the concentration of ciprofloxacin eluted was determined. RESULTS: Ciprofloxacin-treated sewing rings had greater gross tissue incorporation than untreated rings in the presence of a bacterial challenge (P=0.005). Ciprofloxacin-treated rings also had a lower incidence of frank purulence, but this did not reach statistical significance. After 14 days of implantation, ciprofloxacin treated rings had fewer neutrophils (P=0.018) and greater histological tissue incorporation (P=0.017) than untreated rings. The explanted ciprofloxacin-treated rings maintained a zone of inhibition of 3.0+/-1.0 mm after 1 day of implantation and 1.3+/-0.6 mm after 2 days. Ciprofloxacin could be eluted in significant quantities from the explanted rings after 7 days of implantation. CONCLUSIONS: Ciprofloxacin treated polyester can be incorporated into an annuloplasty ring construct that demonstrates excellent tissue incorporation and infection resistance. This study supports the use of this construct in the mitral position in a large animal model.

Development of an infection-resistant bifunctionalized Dacron biomaterial.

A novel infection-resistant biomaterial was created by applying the antibiotic Ciprofloxacin (Cipro) to a recently developed bifunctionalized polyethylene terephthalate ("polyester," Dacron) material using textile-dyeing technology. Dacron was modified via exposure to ethylenediamine (EDA) to create amine and carboxylic acid sites within the polymer backbone. Cipro was applied to the bifunctionalized Dacron construct under varied experimental conditions, with resulting antimicrobial activity determined via zone of inhibition. Dacron segments treated at a liquor ratio of 20:1, with 5% Cipro on weight of fabric (owf), at pH 8 for 4 h at 70 degrees C followed by autoclaving showed antimicrobial activity for 78 days (length of study). Segments treated similarly but without autoclaving lost activity within 1 day. Dyeing time and temperature did not significantly affect antibiotic release/activity, but segments dyed at pHs higher or lower than 8 had less antimicrobial activity. The long-term infection resistance provided by this technique may answer major problems of infection from which implantable Dacron biomedical devices suffer.

Development of an infection-resistant, bioactive wound dressing surface.

Trauma, whether caused by an accident or in an intentional manner, results in significant morbidity and mortality. The goal of this study was to develop a novel biomaterial surface in vitro and ex vivo that provides both localized infection resistance nd hemostatic properties. Our hypothesis is that a combination of specific surface characteristics can be successfully incorporated into a single biomaterial. Functional groups were created with woven Dacron (Cntrl) material via exposure to ethylenediamine (C-EDA). The antibiotic ciprofloxacin (Cipro) was then applied to the C-EDA material using pad/autoclave technique (C-EDA-AB) followed by surface immobilization of the coagulation cascade enzyme thrombin (C-EDA-AB-Thrombin). Antimicrobial activity by the C-EDA-AB surface persisted for 5 days compared with Cntrl and dipped controls, which lasted <1 h. C-EDA-AB-Thrombin surfaces had 2.6- and 105-fold greater surface thrombin activity compared with nonspecifically bound thrombin and Cipro-dyed surfaces, respectively. Surface thrombus formation ex vivo was evident after 1 min of exposure, with thrombus organization evident by 2.5 min. In contrast, C-EDA-AB and Cntrl segments showed only blood protein adsorption on the fibers. Thus, this study demonstrated that Cipro and thrombin can be simultaneously incorporated onto a biomaterial surface while maintaining their respective biological activities.

A biologically active VEGF construct in vitro: implications for bioengineering-improved prosthetic vascular grafts.

Prosthetic arterial grafts are unable to develop an intact endothelial lining after implantation, predisposing them to fail. Strategies have been sought to enhance endothelialization using growth factors and cytokines. This study assessed the biologic activity of vascular endothelial growth factor (VEGF) covalently linked to bovine serum albumin (BSA). Native and modified VEGF were assayed for endothelial cell migration and proliferation. Migration assays were performed comparing the effects of 2% fetal bovine serum (FBS), 50 ng/mL, 100 ng/mL, and 200 ng/mL of native VEGF and VEGF-BSA. Proliferation assays were performed by using Alamar Blue comparing cellular growth in 1% FBS, 10% FBS, 100 ng/mL unbound VEGF, and 100 ng/mL VEGF-BSA. VEGF is a potent chemotactic agent for endothelial cells in both unbound and bound states. Native VEGF solutions (50 ng/mL, 100 ng/mL, and 200 ng/mL) stimulated 23.9 cells/high power field (HPF), 35.3 cells/HPF, and 49.1 cells/HPF (p < 0.005). VEGF-BSA solutions stimulated 25.9 cells/HPF, 39.1 cells/HPF, and 69.0 cells/HPF (p < 0.001). VEGF-BSA and native VEGF supported similar increased cellular proliferation compared with 1% FBS media (p < 0.002). Modified VEGF retains its chemotactic and proliferative properties in vitro. These findings suggest that bare prosthetic surfaces lined with VEGF bound to a "basecoat" albumin may support endothelial cell proliferation and migration and thereby offer new strategies to improve graft patency.