George L. Collins

Neurite extension of primary neurons on electrospun piezoelectric scaffolds

Neural tissue engineering may be a promising option for neural repair treatment, for which a well-designed scaffold is essential. Smart materials that can stimulate neurite extension and outgrowth have been investigated as potential scaffolding materials. A piezoelectric polymer polyvinylidene fluoride-trifluoroethylene (PVDF-TrFE) was used to fabricate electrospun aligned and random scaffolds having nano- or micron-sized fiber dimensions. The advantage of using a piezoelectric polymer is its intrinsic electrical properties. The piezoelectric characteristics of PVDF-TrFE scaffolds were shown to be enhanced by annealing. Dorsal root ganglion (DRG) neurons attached to all fibrous scaffolds. Neurites extended radially on random scaffolds, whereas aligned scaffolds directed neurite outgrowth for all fiber dimensions. Neurite extension was greatest on aligned, annealed PVDF-TrFE having micron-sized fiber dimensions in comparison with annealed and as-spun random PVDF-TrFE scaffolds. DRG on micron-sized aligned, as-spun and annealed PVDF-TrFE also had the lowest aspect ratio amongst all scaffolds, including non-piezoelectric PVDF and collagen-coated substrates. Findings from this study demonstrate the potential use of a piezoelectric fibrous scaffold for neural repair applications.

Functional 3-D Cardiac Co-Culture Model Using Bioactive Chitosan Nanofiber Scaffolds

The in vitro generation of a three‐dimensional (3‐D) myocardial tissue‐like construct employing cells, biomaterials, and biomolecules is a promising strategy in cardiac tissue regeneration, drug testing, and tissue engineering applications. Despite significant progress in this field, current cardiac tissue models are not yet able to stably maintain functional characteristics of cardiomyocytes for long‐term culture and therapeutic purposes. The objective of this study was to fabricate bioactive 3‐D chitosan nanofiber scaffolds using an electrospinning technique and exploring its potential for long‐term cardiac function in the 3‐D co‐culture model. Chitosan is a natural polysaccharide biomaterial that is biocompatible, biodegradable, non‐toxic, and cost effective. Electrospun chitosan was utilized to provide structural scaffolding characterized by scale and architectural resemblance to the extracellular matrix (ECM) in vivo. The chitosan fibers were coated with fibronectin via adsorption in order to enhance cellular adhesion to the fibers and migration into the interfibrous milieu. Ventricular cardiomyocytes were harvested from neonatal rats and studied in various culture conditions (i.e., mono‐ and co‐cultures) for their viability and function. Cellular morphology and functionality were examined using immunofluorescent staining for alpha‐sarcomeric actin (SM‐actin) and gap junction protein, Connexin‐43 (Cx43). Scanning electron microscopy (SEM) and light microscopy were used to investigate cellular morphology, spatial organization, and contractions. Calcium indicator was used to monitor calcium ion flux of beating cardiomyocytes. The results demonstrate that the chitosan nanofibers retained their cylindrical morphology in long‐term cell cultures and exhibited good cellular attachment and spreading in the presence of adhesion molecule, fibronectin. Cardiomyocyte mono‐cultures resulted in loss of cardiomyocyte polarity and islands of non‐coherent contractions. However, the cardiomyocyte‐fibroblast co‐cultures resulted in polarized cardiomyocyte morphology and retained their morphology and function for long‐term culture. The Cx43 expression in the fibroblast co‐culture was higher than the cardiomyocytes mono‐culture and endothelial cells co‐culture. In addition, fibroblast co‐cultures demonstrated synchronized contractions involving large tissue‐like cellular networks. To our knowledge, this is the first attempt to test chitosan nanofiber scaffolds as a 3‐D cardiac co‐culture model. Our results demonstrate that chitosan nanofibers can serve as a potential scaffold that can retain cardiac structure and function. These studies will provide useful information to develop a strategy that allows us to generate engineered 3‐D cardiac tissue constructs using biocompatible and biodegradable chitosan nanofiber scaffolds for many tissue engineering applications. Biotechnol. Bioeng. 2013; 110: 637–647.

Solvent-dependent properties of electrospun fibrous composites for bone tissue regeneration

Biodegradable polymer-ceramic composite scaffolds have gained importance in recent years in the field of orthopedic biomaterials and tissue engineering scaffolds for improving the rate of degradation and limited mechanical properties of bioactive ceramics. This study sought to create composites using the electrospinning process to achieve fibrous scaffolds with uniform fiber morphologies and uniform ceramic dispersions. Composites consisting of 20% hydroxyapatite/80% beta-tricalcium phosphate (20/80 HA/TCP) and poly (epsilon-caprolactone) (PCL) were fabricated. The 20/80 HA/TCP composition was chosen as the ceramic component because of previous reports of greater bone tissue formation in comparison with HA or TCP alone. For electrospinning, PCL was dissolved in either methylene chloride (Composite-MC) or a combination of methylene chloride (80%) and dimethylformamide (20%) (Composite-MC + DMF). Composite-MC mats contained a bimodal distribution of fiber diameters with nanofibers between larger, micron-sized fibers with an average pore size of 79.6 + or - 67 microm, whereas Composite-MC + DMF fibers had uniform fiber diameters with an average pore size of 7.0 + or - 4.2 microm. Elemental mapping determined that the ceramic was distributed throughout the mat and inside the fiber for both composites. However, physical characterization using differential scanning calorimetry (DSC) and mechanical testing revealed that the ceramic in the mats produced with MC + DMF were more uniformly dispersed than the ceramic in the mats produced with MC alone. Maximum tensile stress and strain were significantly higher for Composite-MC + DMF mats compared with Composite-MC mats and were comparable with the mechanical properties of mats of PCL alone. For both composites, there was molecular interaction between the PCL and the ceramic, as demonstrated by a maximum increase of approximately 10 degrees C in the glass transition values with the addition of the ceramic, as confirmed by Fourier transform infrared analysis. In addition, the crystallization behavior of the composites suggested that the ceramic was acting as a nucleating agent. Cell viability studies using human mesenchymal stem cells (MSC) showed that both composite scaffolds supported cell growth. However, cell numbers at early time points in culture were significantly higher on mats produced from MC + DMF compared with mats prepared with MC alone. Further examination revealed that cells were able to infiltrate the pores of the Composite-MC mats, but remained on the outer surface of the Composite-MC + DMF and unfilled PCL mats during the culture period. The results of this study demonstrate that the solvent or solvent combination used in preparing the electrospun composite mats plays a critical role in determining its properties, which may, in turn, affect cell behavior.

Charge generation, charge transport, and residual charge in the electrospinning of polymers: A review of issues and complications

Electrospinning has become a widely implemented technique for the generation of nonwoven mats that are useful in tissue engineering and filter applications. The overriding factor that has contributed to the popularity of this method is the ease with which fibers with submicron diameters can be produced. Fibers on that size scale are comparable to protein filaments that are observed in the extracellular matrix. The apparatus and procedures for conducting electrospinning experiments are ostensibly simple. While it is rarely reported in the literature on this topic, any experience with this method of fiber spinning reveals substantial ambiguities in how the process can be controlled to generate reproducible results. The simplicity of the procedure belies the complexity of the physical processes that determine the electrospinning process dynamics. In this article, three process domains and the physical domain of charge interaction are identified as important in electrospinning: (a) creation of charge carriers...

Thermal analysis of Vectran® fibers and films

Vectra® liquid crystalline polymers (LCPs) were introduced as commercial products in the mid-1980s. The first of these (Vectra A130) was a wholly aromatic thermotropic copolyester ofp-hydroxybenzoic acid and 6-hydroxy-2-naphthoic acid. Vectra A130 is a thermotropic LCP that can be melt spun into filaments that on heat treatment are characterized by high strength and high modulus. Vectra resin can also be extruded into films. In the fiber or film form this material is commercially known as Vectran®. Heat treatment enhances the tensile strength of Vectran fiber variants. Because of this, the elucidation of the physical transformations taking place in the internal structure of the material during heating has always been an important subject. Several thermal techniques are used to indicate clearly that what is observed as a “glass transition” is unlike the conventional glass transition in typical semicrystalline polymers. There is also an indication of the presence of multiple states of mesophase aggregation that collapse into a single state when taken to high enough temperatures.

The Glass Transition and Sub-Tg-Relaxation in Pharmaceutical Powders and Dried Proteins by Thermally Stimulated Current

The main goal of the study was to evaluate the applicability of thermally stimulated current (TSC) as a measure of molecular mobility in dried globular proteins. Three proteins, porcine somatotropin, bovine serum albumin, and immunoglobulin, as well as materials with a strong calorimetric glass transition (T(g)), that is, indomethacin and poly(vinypyrrolidone) (PVP), were studied by both TSC and differential scanning calorimetry (DSC). Protein/sugar colyophilized mixtures were also studied by DSC, to estimate calorimetric T(g) for proteins using extrapolation procedure. In the majority of cases, TSC detected relaxation events that were not observed by DSC. For example, a sub-T(g) TSC event (beta-relaxation) was observed for PVP at approximately 120 degrees C, which was not detected by the DSC. Similarly, DSC did not detect events in any of the three proteins below the thermal denaturation temperature whereas a dipole relaxation was detected by TSC in the range of 90-140 degrees C depending on the protein studied. The TSC signal in proteins was tentatively assigned as localized mobility of protein segments, which is different from a large-scale cooperative motions usually associated with calorimetric T(g). TSC is a promising method to study the molecular mobility in proteins and other materials with weak calorimetric T(g).

Controlled formation of cross-linked collagen fibers for neural tissue engineering applications.

Fibrous scaffolds engineered to direct the growth of tissues can be important in forming architecturally functional tissue such as aligning regenerating nerves with their target. Collagen is a commonly used substrate used for neuronal growth applications in the form of surface coatings and hydrogels. The wet spinning technique can create collagen fibers without the use of organic solvents and is typically accomplished by extruding a collagen dispersion into a coagulation bath. To create well-controlled and uniform collagen fibers, we developed an automatic wet spinning device with precise control over the spinning and fiber collection parameters. A fiber collection belt allowed the continuous formation of very soft and delicate fibers up to half a meter in length. Wet-spun collagen fibers were characterized by tensile and thermal behavior, diameter uniformity, the swelling response in phosphate buffered saline and their biocompatibility with dorsal root ganglion (DRG) neurons and Schwann cells. Fibers formed from 0.75% weight by volume (w/v) collagen dispersions formed the best fibers in terms of tensile behavior and fiber uniformity. Fibers post-treated with the cross-linkers glutaraldehyde and genipin exhibited increased mechanical stability and reduced swelling. Importantly, genipin-treated fibers were conducive to DRG neurons and Schwann cell survival and growth, which validated the use of this cross-linker for neural tissue engineering applications.

Process–structure–property relationships of erodable polymeric biomaterials: II—long range order in poly(desaminotyrosyl arylates)

Abstract The long-range order of some bioerodable polyesteramides based on a desaminotyrosyl [Thermochim Acta 396 (2003) 141; Polym Adv Technol 13 (2002) 926; J Am Chem Soc 119 (1997) 4553] diol monomer has been investigated. The order is mesogenic, best described as a ‘condis crystal’ or smectic-like. In all cases where long-range order is present, ordered H bonds between amide groups are observed. The order stabilizes the polymer to dimensional change and mechanical relaxation under biorelevant conditions.

Thermally stimulated currents observed in pharmaceutical products

Abstract Stability and processing of pharmaceutical products are often related to the molecular mobility of the formulation. It is crucial to know all the transitions or relaxations of the products. Differential scanning calorimetry (DSC) measurements give valuable information about the melt of the substances. The challenge is actually to detect very weak glass transitions and even beta relaxations. The aim of this presentation is to show that thermally stimulated current (TSC) is a driven technique that provides a better sensitivity to glass transition and sub-glass relaxations. TSC was applied to the evaluation of the stress in tablets and on the measurement of low amorphous content (in the order of 2%). TSC complements DSC to obtain the relaxation/transition “spectrum” of a pharmaceutical product.

Evaluating protein incorporation and release in electrospun composite scaffolds for bone tissue engineering applications

Electrospun polymer/ceramic composites have gained interest for use as scaffolds for bone tissue engineering applications. In this study, we investigated methods to incorporate Platelet Derived Growth Factor-BB (PDGF-BB) in electrospun polycaprolactone (PCL) or PCL prepared with polyethylene oxide (PEO), where both contained varying levels (up to 30 wt %) of ceramic composed of biphasic calcium phosphates, hydroxyapatite (HA)/β-tricalcium phosphate (TCP). Using a model protein, lysozyme, we compared two methods of protein incorporation, adsorption and emulsion electrospinning. Adsorption of lysozyme on scaffolds with ceramic resulted in minimal release of lysozyme over time. Using emulsion electrospinning, lysozyme released from scaffolds containing a high concentration of ceramic where the majority of the release occurred at later time points. We investigated the effect of reducing the electrostatic interaction between the protein and the ceramic on protein release with the addition of the cationic surfactant, cetyl trimethylammonium bromide (CTAB). In vitro release studies demonstrated that electrospun scaffolds prepared with CTAB released more lysozyme or PDGF-BB compared with scaffolds without the cationic surfactant. Human mesenchymal stem cells (MSCs) on composite scaffolds containing PDGF-BB incorporated through emulsion electrospinning expressed higher levels of osteogenic markers compared to scaffolds without PDGF-BB, indicating that the bioactivity of the growth factor was maintained. This study revealed methods for incorporating growth factors in polymer/ceramic scaffolds to promote osteoinduction and thereby facilitate bone regeneration.