Poly(ester-ether)s: III. assessment of cell behaviour on nanofibrous scaffolds of PCL, PLLA and PDX blended with amorphous PMeDX.

The aim of this paper is to investigate the physico-chemical properties, degradation behaviour and cellular response of electrospun fibre-scaffolds of semi-crystalline PCL, PLLA and PDX blended with amorphous poly(methyl dioxanone) (PMeDX). Electrospun PCL/PMeDX and PLLA/PMeDX blend mats in varying weight ratios of the two components were fabricated and their overall performance was compared with similar composition PDX/PMeDX scaffolds. DSC analysis showed almost no change in crystallization temperature of PCL with increasing PMeDX content and TGA showed a different degradation profile as PMeDX content increased. The appearance of two crystallization peaks for PLLA/PMeDX blends suggested stereocomplex formation. As noted from AFM images, addition of PMeDX caused a change in the width of the lamellae from 14.8 ± 2.9 nm in 100/0 mat to 32.0 ± 11.5 nm in 85/15 mat. Moreover, PCL/PMeDX blend mats show a significant drop in Young's modulus for 93/7, 90/10 and 85/15 compositions compared to 100/0 and 98/2. On the other hand, no clear trend in mechanical properties was observed for espun PLLA/PMeDX mats with increasing PMeDX content. Based on these analyses, it was concluded that PCL and PMeDX were immiscible while miscible blends were obtained with PLLA and PMeDX. Initial degradation of electrospun mats over a period of 5 weeks appears to occur via a surface erosion mechanism. In vitro cell culture studies using HDFs showed that the scaffolds were bioactive and a greater density of viable cells was noted on electrospun PCL/PMeDX and PLLA/PMeDX scaffolds compared to PCL and PLLA mats respectively. HDFs infiltrated through the entire thickness of espun 85/15 PLLA/PMeDX scaffold due to a combination of factors including morphology, porosity, surface characteristics and mechanical properties.

Modulation of mast cell adhesion, proliferation, and cytokine secretion on electrospun bioresorbable vascular grafts.

Mast cells synthesize several potent angiogenic factors and can also stimulate fibroblasts, endothelial cells, and macrophages. An understanding of how they participate in wound healing and angiogenesis is important to further our knowledge about in situ vascular prosthetic regeneration. The adhesion, proliferation, and cytokine secretion of bone marrow-derived murine mast cells (BMMC) on electrospun polydioxanone, polycaprolactone, and silk scaffolds, as well as tissue culture plastic, has been investigated in the presence or absence of IL-3, stem cell factor, IgE and IgE with a crosslinking antigen, dinitrophenol-conjugated albumin (DNP). It was previously believed that only activated BMMCs exhibit adhesion and cytokine secretion. However, this study shows nonactivated BMMC adhesion to electrospun scaffolds. Silk scaffold was not found to be conducive for mast cell adhesion and cytokine secretion. Activation by IgE and DNP significantly enhanced mast cell adhesion, proliferation, migration, and secretion of tumor necrosis factor alpha, macrophage inflammatory protein-1α, and IL-13. This indicates that mast cells might play a role in the process of biomaterial integration into the host tissue, regeneration, and possibly angiogenesis.

Preliminary investigation of airgap electrospun silk-fibroin-based structures for ligament analogue engineering.

The process of electrospinning has proven to be highly beneficial for use in a number of tissue-engineering applications due to its ease of use, flexibility and tailorable properties. There have been many publications on the creation of aligned fibrous structures created through various forms of electrospinning, most involving the use of a metal target rotating at high speeds. This work focuses on the use of a variation known as airgap electrospinning, which does not use a metal collecting target but rather a pair of grounded electrodes equidistant from the charged polymer solution to create highly aligned 3D structures. This study involved a preliminary investigation and comparison of traditionally and airgap electrospun silk-fibroin-based ligament constructs. Structures were characterized with SEM and alignment FFT, and underwent porosity, permeability, and mechanical anisotropy evaluation. Preliminary cell culture with human dermal fibroblasts was performed to determine the degree of cellular orientation and penetration. Results showed airgap electrospun structures to be anisotropic with significantly increased porosity and cellular penetration compared to their traditionally electrospun counterparts.

Electrospinning-aligned and random polydioxanone-polycaprolactone-silk fibroin-blended scaffolds: geometry for a vascular matrix.

Extracellular matrices are arranged with a specific geometry based on tissue type and mechanical stimulus. For blood vessels in the body, preferential alignment of fibers is in the direction of repetitive force. Electrospinning is a controllable process which can result in fiber alignment and randomization depending on the parameters utilized. In this study, arterial grafts composed of polycaprolactone (PCL), polydioxanone (PDO) and silk fibroin in blends of 100:0 and 50:50 for both PCL:silk and PDO:silk were investigated to determine if fibers could be controllably aligned using a mandrel rotational speed ranging from 500 to 8000 revolutions per minute (RPM). Results revealed that large- and small-diameter mandrels produced different degrees of fiber alignment based on a fast Fourier transform of scanning electron microscope images. Uniaxial tensile testing further demonstrated scaffold anisotropy through changes in peak stress, modulus and strain at break at mandrel rotational speeds of 500 and 8000 RPM, causing peak stress and modulus for PCL to increase 5- and 4.5-fold, respectively, as rotational speed increased. Additional mechanical testing was performed on grafts using dynamic compliance, burst strength and longitudinal strength displaying that grafts electrospun at higher rotational rates produced stiffer conduits which had lower compliance and higher burst strength compared to the lower mandrel rotational rate. Scaffold properties were found to depend on several parameters in the electrospinning process: mandrel rotational rate, polymer type, and mandrel size. Vascular scaffold design under anisotropic conditions provided interesting insights and warrants further investigation.

Angiogenic potential of human macrophages on electrospun bioresorbable vascular grafts.

The aim of this study was to investigate macrophage interactions with electrospun scaffolds and quantify the expression of key angiogenic growth factors in vitro. This study will further help in evaluating the potential of these electrospun constructs as vascular grafts for tissue repair and regeneration in situ. Human peripheral blood macrophages were seeded in serum free media on electrospun (10 mm) discs of polydioxanone (PDO), elastin and PDO:elastin blends (50:50, 70:30 and 90:10). The growth factor secretion was analyzed by ELISA. Macrophages produced high levels of vascular endothelial growth factor and acidic fibroblast growth factor. Transforming growth factor beta-1 (TGF-beta1) secretion was relatively low and there was negligible production of basic fibroblast growth factor. Therefore, it can be anticipated that these scaffolds will support tissue regeneration and angiogenesis.

Electrospun polydioxanone-elastin blends: potential for bioresorbable vascular grafts.

An electrospun cardiovascular graft composed of polydioxanone (PDO) and elastin has been designed and fabricated with mechanical properties to more closely match those of native arterial tissue, while remaining conducive to tissue regeneration. PDO was chosen to provide mechanical integrity to the prosthetic, while elastin provides elasticity and bioactivity (to promote regeneration in vitro/in situ). It is the elastic nature of elastin that dominates the low-strain mechanical response of the vessel to blood flow and prevents pulsatile energy from being dissipated as heat. Uniaxial tensile and suture retention tests were performed on the electrospun grafts to demonstrate the similarities of the mechanical properties between the grafts and native vessel. Dynamic compliance measurements produced values that ranged from 1.2 to 5.6%/100 mmHg for a set of three different mean arterial pressures. Results showed the 50:50 ratio to closely mimic the compliance of native femoral artery, while grafts that contained less elastin exceeded the suture retention strength of native vessel. Preliminary cell culture studies showed the elastin-containing grafts to be bioactive as cells migrated through their full thickness within 7 days, but failed to migrate into pure PDO scaffolds. Electrospinning of the PDO and elastin-blended composite into a conduit for use as a small diameter vascular graft has extreme potential and warrants further investigation as it thus far compares favorably to native vessel.

Regulation of cellular infiltration into tissue engineering scaffolds composed of submicron diameter fibrils produced by electrospinning.

We characterize the infiltration of interstitial cells into tissue engineering scaffolds prepared with electrospun collagen, electrospun gelatin, electrospun poly(glycolic) acid (PGA), electrospun poly(lactic) acid (PLA), and an electrospun PGA/PLA co-polymer. Electrospinning conditions were optimized to produce non-woven tissue engineering scaffolds composed of individual fibrils less than 1000 nm in diameter. Each of these materials was then electrospun into a cylindrical construct with a 2 mm inside diameter with a wall thickness of 200-250 microm. Electrospun scaffolds of collagen were rapidly, and densely, infiltrated by interstitial and endothelial cells when implanted into the interstitial space of the rat vastus lateralis muscle. Functional blood vessels were evident within 7 days. In contrast, implants composed of electrospun gelatin or the bio-resorbable synthetic polymers were not infiltrated to any great extent and induced fibrosis. Our data suggests that topographical features, unique to the electrospun collagen fibril, promote cell migration and capillary formation.

The persistence of electrostatically seeded endothelial cells lining a small diameter expanded polytetrafluoroethylene vascular graft.

PURPOSE: The purpose of this study was to evaluate the persistence of electrostatically seeded endothelial cells (ECs) lining an expanded polytetrafluorethylene (e-PTFE) graft after one week exposure to in vivo circulation in a canine femoral artery bypass model. This was accomplished by visualizing the PKH 26 (red fluorescent) label placed in the EC membranes prior to the seeding procedure. Furthermore, this study was performed to confirm that the source of the ECs lining the graft were those from the initial inoculum. METHODS: This evaluation consisted of harvesting autologous, canine jugular vein ECs, PKH 26 labeling of the ECs, electrostatic EC seeding the e-PTFE grafts (4mm GORE-TEX, Length=6cm), implanting the grafts (femoral artery model) for one week, and explanting the grafts for light, fluorescent and scanning electron microscopy evaluations of the luminal surface. RESULTS: The unseeded grafts (controls) had a mean fluorescence surface coverage of 6.82 +/- 7.19%, while the EC seeded grafts had a mean of 90.3 +/- 14.3% which is significantly (p <0.001) different from the controls. Overall, the seeding time including the EC harvesting and PKH 26 labeling protocol was approximately 75 min. CONCLUSIONS: The electrostatically seeded ECs persisted after implantation of the graft as demonstrated by the PKH 26 labeling data. The fluorescent data also demonstrated that the neointima formed (EC luminal surface coverage) one week after implantation was in fact derived from the ECs initially seeded as determined by the abundance of the labeled ECs.

Determination of the prime electrostatic endothelial cell transplantation procedure for e-PTFE vascular prostheses.

The purpose of this study was to evaluate the extent of cellular adhesion (density and morphological maturation), cellular membrane damage, and cellular viability after an electrostatic transplantation of human umbilical vein endothelial cells (HUVECs) onto 6-cm segments of 4-mm I.D. e-PTFE (GORE-TEX) vascular prostheses using a prototype electrostatic endothelial cell transplantation device (EECTD). The electrostatic transplantation parameters evaluated were the apparatus-applied voltage and transplantation time. By our definition, the combination of applied voltage and transplantation time that met the a priori criteria of: 1) maximum transplanted cellular viability, 2) maximum transplantation density, 3) maximum morphological maturation (degree of cellular flattening), and 4) minimal cellular membrane damage would be the prime transplantation procedure. The results of the experimentation indicated that the prime conditions for HUVEC transplantation were obtained when +1.0 V was applied for a transplantation time of 16 min. These conditions achieved an average viable graft surface coverage of 97.4+/-1.6% with an average transplantation density of 73,540+/-8.514 HUVECs/cm2. Furthermore, the transplanted HUVECs were morphologically mature (flattened) with minimal apparent cellular membrane damage (lysis or pitting). The overall clinical significance of this study is that viable endothelial cell transplantation to synthetic vascular grafts can be accomplished at high cellular densities and morphological maturation in 16 min using the EECTD. With the promising in vitro transplantation results, the next logical investigations will include additional in vitro evaluations (cellular retention upon shear stress exposure and biochemical assays) followed by in vivo evaluations to examine thromboresistance and influence on intimal/anastomotic hyperplasia.

In vitro evaluation of electrostatic endothelial cell transplantation onto 4 mm interior diameter expanded polytetrafluoroethylene grafts.

PURPOSE: To perform an in vitro evaluation of electrostatic endothelial cell transplantation of human umbilical vein endothelial cells (HUVEC) onto segments of 4 mm internal diameter expanded polytetrafluoroethylene (ePTFE) vascular prostheses. METHODS: This evaluation consisted of exposing vascular graft segments that had been subjected to either electrostatic or gravitation transplantation with HUVEC to a physiologic shear stress (15 dynes/cm2) under steady flow conditions within a flow loop system. Biochemical assays were performed on freshly transplanted grafts by means of radioimmunoassay for prostacyclin and thromboxane A2. RESULTS: There was a 30% loss of HUVEC after 30 minutes of shear stress exposure from the grafts subjected to gravitational transplantation with no additional significant (alpha = 0.05) loss after 120 minutes. Grafts subjected to electrostatic transplantation had no significant (alpha = 0.05) loss of HUVEC during exposure to physiologic shear stress. Furthermore, after 120 minutes of shear-stress exposure, the grafts subjected to electrostatic transplantation (78,420 +/- 6274 HUVEC/cm2) retained 2.3 times more HUVEC than the counterparts subjected to gravitational transplantation (34,427 +/- 4637 HUVEC/cm2). The biochemical assay results indicated no significant (alpha = 0.05) production of prostacyclin or thromboxane A2 regardless of the method of cell transplantation. CONCLUSIONS: (1) The electrostatic transplantation technique was superior to the gravitational transplantation technique in terms of cellular retention when the ePTFE grafts were exposed to physiologic shear stress. (2) Production of prostacyclin and thromboxane A2 did not differ between transplanted HUVEC subjected to gravitational or electrostatic procedures.

Electrostatic endothelial cell seeding technique for small-diameter (<6 mm) vascular prostheses: feasibility testing.

Multiple studies have indicated the importance of surface charge in the adhesion of multiple cardiovascular cell lines including platelets and endothelial cells on the substrate materials (1,4,7-10,12-15). It is the purpose of this article to report a feasibility study conducted using an electrostatic endothelial cell seeding technique. The feasibility study was conducted using human umbilical vein endothelial cells (HUVEC), a static pool apparatus, a voltage source, and a parallel plate capacitor. The HUVEC concentration and seeding times were constant at 560,000 HUVEC/ml and 30 min, respectively. Scanning electron microscopy examination of the endothelial cell adhesion indicated that an induced temporary positive surface charge on e-PTFE graft material enhances the number and the maturation (flattening) of HUVECs adhered. The results indicated that the total number of endothelial cells adhered (70.9 mm2) was increased from 9198 +/- 1194 HUVECs on the control (no induced surface charge) e-PTFE to 22,482 +/- 4814 HUVECs (2.4 x control) on the maximum induced positive surface charge. The total number of cells in the flattened phase of adhesion increased from 837 +/- 275 to 6785 +/- 1012 HUVECs (8.1x) under identical conditions. Thus, the results of the feasibility study support the premise that electrostatic interaction is an important factor in both the endothelial cell adhesion and spreading processes and suggest that the electrostatic seeding technique may lead to an increased patency of small diameter (<6 mm) vascular prostheses.

Electrostatic endothelial cell transplantation within small-diameter (<6 mm) vascular prostheses: a prototype apparatus and procedure.

This article presents a novel, clinically relevant electrostatic endothelial cell transplantation (seeding/sodding) device (U.S. & Foreign Patent Protections Pending) for small-diameter (<6 mm) vascular prostheses. The prototype apparatus was designed and built to tissue engineer 4.0 mm, I.D. GORE-TEX (W.L. Gore & Associates, Inc.) standard wall graft segments varying in length from 4 to 12 cm. The prototype electrostatic endothelial cell transplantation apparatus is composed of an external and internal conductor, aluminum base, end supports, pillow blocks, filling apparatus, electric motor drive system, and a voltage source. The cylindrical capacitor arrangement of the device along with an electrical potential applied across the internal and external conductors creates the unique feature of this endothelial cell transplantation technique, an electric field within the cylindrical capacitor (within the graft lumen) which in turn induces a temporary positive surface charge on the graft (dielectric material) luminal surface. Multiple studies have shown that a positively charged substrate is more conducive to endothelial cell adhesion and morphological maturation (flattening) (1,2, 7,8,10,13-15). This induced positive surface charge dissipates immediately upon removal from the electrostatic endothelial cell transplantation device. Thus, after endothelial cell adhesion the graft luminal surface reverts back to its natural (nonthrombogenic) negative surface charge.