Robert F. Valentini

RGD-coated titanium implants stimulate increased bone formation in vivo

Numerous studies have demonstrated that peptide modified surfaces influence short- and long-term cell responses such as attachment, shape and function in vitro. These responses are mediated via cell receptors known as integrins which bind specifically to short peptide sequences from larger proteins. Integrins transduce information to the nucleus through several cytoplasmic signalling pathways. Little is known, however, about the ability of peptide-coated surfaces to influence cell responses in vivo. The present study was designed to evaluate the quality and quantity of the new bone formed in response to titanium rods surface-coated with the peptide sequence Arg-Gly-Asp-Cys (RGDC) using gold-thiol chemistry and implanted in rat femurs. Histomorphometric analysis of cross-sections perpendicular to the implant long axis showed a significantly thicker shell of new bone formed around RGD-modified versus plain implants at 2 weeks (26.2 +/- 1.9 vs. 20.5 +/- 2.9 microm; P < 0.01). A significant increase in bone thickness for RGD implants was also observed at 4 weeks while bone surrounding controls did not change significantly in thickness (32.7 +/- 4.6 vs. 22.6 +/- 4.0 microm; P < 0.02). Mechanical pull-out testing conducted at 4 weeks revealed the average interfacial shear strength of peptide modified rods was 38% greater than control rods although this difference was not statistically significant. These pilot data suggest that an RGDC peptide coating may enhance titanium rod osseointegration in the rat femur. Long-term studies and evaluation of other peptides in larger animal models are warranted.

Drug‐screening platform based on the contractility of tissue‐engineered muscle

A tissue‐based approach to in vitro drug screening allows for determination of the cumulative positive and negative effects of a drug at the tissue rather than the cellular or subcellular level. Skeletal muscle myoblasts were tissue‐engineered into three‐dimensional muscle with parallel myofibers generating directed forces. When grown attached to two flexible microposts (μposts) acting as artificial tendons in a 96‐well plate format, the miniature bioartificial muscles (mBAMs) generated tetanic (active) forces upon electrical stimulation measured with a novel image‐based motion detection system. mBAM myofiber hypertrophy and active force increased in response to insulin‐like growth factor 1. In contrast, mBAM deterioration and weakness was observed with a cholesterol‐lowering statin. The results described in this study demonstrate the integration of tissue engineering and biomechanical testing into a single platform for the screening of compounds affecting muscle strength. Muscle Nerve, 2007

Collagen- and laminin-containing gels impede peripheral nerve regeneration through semipermeable nerve guidance channels.

Semipermeable guidance channels were filled with saline, collagen-, or laminin-containing gels and used to repair a 4-mm sciatic nerve gap in mice. After 12 weeks, nerve cables regenerated in gel-filled channels displayed fewer myelinated axons than saline-filled channels. Remnants of the exogenous substrates were still in evidence, in amounts related to the initial collagen or laminin gel concentration. The impairment of nerve regeneration by collagen or laminin-containing gels suggests that the regenerative environment created within semipermeable channels is not improved by the addition of growth substrates in a gel form.

Electrically charged polymeric substrates enhance nerve fibre outgrowth In vitro

The physical, chemical and electrical properties of synthetic guidance devices are known to influence nerve regeneration in vivo. In the present study, neurons were cultured directly on electrically charged polymer growth substrates to determine if local electrical charges enhance nerve fibre outgrowth in vitro. Piezoelectric polymers such as polyvinylidene fluoride (PVDF) generate transient surface charges under minute mechanical strain. Mouse neuroblastoma (Nb2a) cells were cultured directly on electrically poled (i.e. piezoelectric) and unpoled (i.e. nonpiezoelectric) PVDF substrates in serum-free and serum-containing media. Nerve fibre outgrowth was analysed 24, 48, 72 and 96 h after plating. Piezoelectric PVDF substrates generated 2-3 mV at 1200 Hz when placed on standard incubator shelves and unpoled PVDF substrates showed no output. Nb2a cells grown on piezoelectric substrates exhibited significantly greater levels of process outgrowth and neurite lengths at all time periods for both media conditions. Detailed surface characterization of PVDF substrates using electron spectroscopy for chemical analysis (ESCA) and a comprehensive wettability profile revealed that poled and unpoled PVDF was chemically indistinguishable and showed similar surface wettabilities and adhesive properties. Therefore, we conclude that enhanced process outgrowth was induced by the films piezoelectric output, making poled PVDF a unique biomaterial for which cell/polymer interactions are mediated predominantly through bulk electrical properties rather than surface properties.

PIEZOELECTRIC GUIDANCE CHANNELS ENHANCE REGENERATION IN THE MOUSE SCIATIC NERVE AFTER AXOTOMY

Piezoelectric nerve guidance channels made of polyvinylidene fluoride (PVDF) were evaluated in a transected mouse sciatic nerve model. Poled PVDF channels were compared to unpoled PVDF channels after 4 and 12 weeks of implantation. In all animals, the proximal and distal nerve stumps were bridged by a continuous nerve cable. Nerves regenerated in poled channels contained a higher number of myelinated axons than those regenerated in unpoled channels at both time periods. We conclude that piezoelectric nerve guidance channels enhance peripheral nerve regeneration and provide a tool to investigate the influence of electrical activity on nerve regeneration.

The morphology of regenerating peripheral nerves is modulated by the surface microgeometry of polymeric guidance channels

The present study was designed to evaluate the influence of synthetic guidance channel surface microgeometry on morphological patterns of neural regeneration. Tubes with smooth (S), rough (R), or alternating smooth-rough (S/R) or rough-smooth (R/S) inner surfaces but with identical chemical composition and permeability characteristics were used to bridge a 4-mm nerve gap in a transected mouse sciatic nerve. Animals received S and R channels for 1, 2 and 4 weeks and both S/R and R/S channels for 2 and 4 weeks. At 1 week, the S tubes contained a longitudinally oriented fibrin matrix not contacting the channels smooth inner wall, whereas R tubes featured an unorganized fibrin matrix which, together with fibroblasts and macrophages, had invaded the channels rough trabecular network. After 4 weeks, S tubes contained discrete, free-floating nerve cables with numerous myelinated and unmyelinated axons surrounded by a thin, continuous epineurial-like layer, whereas R tubes were completely filled with a loose connective tissue stroma with only a few axons. In combined S/R or R/S channels, the general morphological patterns in individual S or R segments were similar to those observed in pure S or R channels, regardless of whether the tube segment was positioned at the proximal or distal nerve end. Proximal smooth channel segments contained discrete cables which abruptly fanned out to completely fill the lumen in distal rough segments. The opposite pattern was observed with proximal rough and distal smooth segments. At 4 weeks, myelinated axons were observed along the entire length of S/R and R/S tubes. These results suggest that the surface microgeometry of guidance channels influences the outcome of peripheral nerve regeneration, potentially by affecting the early arrangement of the fibrin matrix and/or inducing different cellular responses.

Improved nerve regeneration through piezoelectric vinylidenefluoride-trifluoroethylene copolymer guidance channels

Piezoelectric materials generating electrical charges in response to mechanical strain may be used to stimulate axonal regeneration following nerve injury. Tubular nerve guidance channels were extruded from a vinylidenefluoride-trifluoroethylene copolymer using a melt-extrusion process. Unlike vinylidenefluoride homopolymer, the copolymer does not need mechanical stretching to achieve a dipole-containing crystal structure, enabling the fabrication of complex piezoelectric devices. Selected tubes were rendered piezoelectric in a high voltage corona poling apparatus. Crystal structure changes induced by poling were evaluated with differential scanning calorimetry. In contrast to unpoled samples, poled ones displayed a sharp endothermic peak and a greater heat of transition at the Curie temperature, indicative of an increase in crystal order and size. The piezoelectric output of poled tubes was characterized using a laser-monitored deflection system interfaced with a charge amplifier and oscilloscope. Poled tubes generated significant voltages in response to slight mechanical deformations. The magnitude of electrical output was independent of the poling polarity. Unpoled tubes showed no electrical output. Positive, negative and unpoled vinylidenefluoride-trifluoroethylene copolymer tubes were used to repair a 10 mm gap in transected sciatic nerves of adult rats. Nerves regenerated in positively poled channels had a significantly greater number of myelinated axons than those regenerated in unpoled channels 4 wk post-implantation. Negatively poled channels contained an intermediate number of myelinated axons. We concluded that piezoelectrically active vinylidenefluoride-trifluoroethylene copolymer tubes significantly enhance nerve regeneration as compared to chemically identical, unpoled tubes and that the polarity of the corona poling procedure used to fabricate piezoelectric materials may play a role in determining biological responses.

Induction of tumor-specific protective immunity by in situ Langerhans cell vaccine

Although anti-tumor immunity is inducible by dendritic cell (DC)–based vaccines, time- and cost-consuming “customizing” processes required for ex vivo DC manipulation have hindered broader clinical applications of this concept. Epidermal Langerhans cells (LCs) migrate to draining lymph nodes and undergo maturational changes on exposure to reactive haptens. We entrapped these migratory LCs by subcutaneous implantation of ethylene–vinyl–acetate (EVA) polymer rods releasing macrophage inflammatory protein (MIP)-3β (to create an artificial gradient of an LC-attracting chemokine) and topical application of hapten (to trigger LC emigration from epidermis). The entrapped LCs were antigen-loaded in situ by co-implantation of the second EVA rods releasing tumor-associated antigens (TAAs). Potent cytotoxic T-lymphocyte (CTL) activities and protective immunity against tumors were induced efficiently with each of three tested TAA preparations. Thus, tumor-specific immunity is inducible by the combination of LC entrapment and in situ LC loading technologies. Our new vaccine strategy requires no ex vivo DC manipulation and thus may provide time and cost savings.

Polymer electret guidance channels enhance peripheral nerve regeneration in mice

Polytetrafluoroethylene (PTFE) tubes were prepared as electrets displaying a quasi-permanent surface charge due to the presence of trapped monopolar charge carriers. PTFE tubes containing either positive or negative charges and electrically neutral PTFE tubes were used as nerve guidance channels for the repair of a 4 mm nerve gap in the sciatic nerve of mice. After 4 weeks of implantation, positively and negatively charged PTFE electrets contained regenerated nerves with significantly more myelinated axons than nerves regenerated in uncharged PTFE tubes. This observation suggests that peripheral nerve regeneration can be enhanced by electrically charged nerve guidance channels.

Influence of surface texture of polymeric sheets on peripheral nerve regeneration in a two-compartment guidance system

Synthetic guidance channels are useful tools to study the mechanisms underlying peripheral nerve regeneration. In the present study, the lumen of silicone elastomer tubes was divided into two compartments by a polymer strip 10 mm long placed along the tube length. The influence of varying the surface texture of hydrophilic and hydrophobic polymer strips on the morphology of the regenerated neural tissue was analysed. Hydrophilic nitrocellulose (NC) and hydrophobic polyvinylidene fluoride (PVDF) films with smooth (S-NC and S-PVDF) or rough (R-NC, R-PVDF) surface texture were used. Five channels of each type were used to repair transected rat sciatic nerves and analysed after 4 wk. Tissue strips bridged the nerve stumps in all R-NC and R-PVDF tubes, in five of the S-NC and three of the S-PVDF tubes. In R-NC and R-PVDF tubes, bell-shaped tissue adhering to the polymer strip was observed, whereas in S-NC and S-PVDF tubes round, free-floating nerve cables were seen. All the cables contained myelinated and unmyelinated axons and Schwann cells grouped in microfascicles and surrounded by an epineurial layer. For both rough strips, the initial cell layer consisted of macrophages adhering to the polymer surface. The epineurial nerve tissue contacting the rough surface was significantly thinner for PVDF compared with NC strips. No difference in epineurial thickness was observed for nerves facing the silicone tube or for smooth NC and PVDF strips. S-PVDF tubes contained significantly more myelinated axons than S-NC tubes.(ABSTRACT TRUNCATED AT 250 WORDS)