Permeable guidance channels containing microfilament scaffolds enhance axon growth and maturation.

Successful peripheral nerve regeneration is still limited in artificial conduits, especially for long lesion gaps. In this study, porous poly(L-lactide-co-DL-lactide, 75:25) (PLA) conduits were manufactured with 16 poly(L-lactide) (PLLA) microfilaments aligned inside the lumen. Fourteen and 18 mm lesion gaps were created in a rat sciatic nerve lesion model. To evaluate the combined effect of permeable PLA conduits and microfilament bundles on axon growth, four types of implants were tested for each lesion gap: PLA conduits with 16 filaments; PLA conduits without filaments; silicone conduits with 16 filaments; and silicone conduits without filaments. Ten weeks following implantation, regeneration within the distal nerve was compared between corresponding groups. Antibodies against the markers S100, calcitonin gene related peptide (CGRP), RMDO95, and P0 were used to identify Schwann cells, unmyelinated axons, myelinated axons, and myelin, respectively. Results demonstrated that the filament scaffold enhanced tissue cable formation and Schwann cell migration in all groups. The filament scaffold enhanced axonal regeneration toward the distal stump, especially across long lesion gaps, but significance was only achieved with PLA conduits. When compared to corresponding silicone conduits, permeable PLA conduits enhanced myelinated axon regeneration across both lesion gaps and achieved significance only in combination with filament scaffolds. Myelin staining indicated PLA conduits supported axon myelination with better myelin quantity and quality when compared to silicone conduits.

Mechanical properties and in vitro degradation of bioresorbable fibers and expandable fiber-based stents.

Bioresorbable polymeric support devices (stents) are being developed in order to improve the biocompatibility and drug reservoir capacity of metal stents, as well as to offer a temporary alternative to permanent metallic stents. These temporary devices may be utilized for coronary, urethral, tracheal, and other applications. The present study focuses on the mechanical properties of bioresorbable fibers as well as stents developed from these fibers. Fibers made of poly(L-lactide) (PLLA), polydioxanone (PDS), and poly(glycolide-co-epsilon-caprolactone) (PGACL) were studied in vitro. These fibers combine a relatively high initial strength and modulus together with sufficient ductility and flexibility, and were therefore chosen for use in stents. The effect of degradation on the tensile mechanical properties and morphology of these fibers was examined. The expandable stents developed from these fibers demonstrated excellent initial radial compression strength. The PLLA stents exhibited excellent in vitro degradation resistance and can therefore support body conduits such as blood vessels for prolonged periods of time. PDS and PGACL stents can afford good support for 5 and 2 weeks, respectively, and can therefore be utilized for short-term applications. The degradation resistance of the stents correlates with the profile of mechanical property deterioration of the corresponding bioresorbable fibers.

Functional repair after dorsal root rhizotomy using nerve conduits and neurotrophic molecules.

Functional recovery after large excision of dorsal roots is absent because of both the limited regeneration capacity of the transected root, and the inability of regenerating sensory fibers to traverse the dorsal root entry zone. In this study, bioresorbable guidance conduits were used to repair 6-mm dorsal root lesion gaps in rats, while neurotrophin-encoding adenoviruses were used to elicit regeneration into the spinal cord. Polyester conduits with or without microfilament bundles were implanted between the transected ends of lumbar dorsal roots. Four weeks later, adenoviruses encoding NGF or GFP were injected into the spinal cord along the entry zone of the damaged dorsal roots. Eight weeks after injury, nerve regeneration was observed through both types of implants, but those containing microfilaments supported more robust regeneration of calcitonin gene-related peptide (CGRP)-positive nociceptive axons. NGF overexpression induced extensive regeneration of CGRP(+) fibers into the spinal cord from implants showing nerve repair. Animals that received conduits containing microfilaments combined with spinal NGF virus injections showed the greatest recovery in nociceptive function, approaching a normal level by 7-8 weeks. This recovery was reversed by recutting the dorsal root through the centre of the conduit, demonstrating that regeneration through the implant, and not sprouting of intact spinal fibers, restored sensory function. This study demonstrates that a combination of PNS guidance conduits and CNS neurotrophin therapy can promote regeneration and restoration of sensory function after severe dorsal root injury.

Protein-coated poly(L-lactic acid) fibers provide a substrate for differentiation of human skeletal muscle cells.

Tissue engineering represents a potential method for repairing damaged skeletal muscle tissue. Extracellular matrix (ECM) proteins were evaluated for their ability to aid in cell attachment, whereas a poly(L-lactic acid) (PLLA) fiber scaffold was tested as a substrate for the differentiation of human skeletal muscle cells. In comparison to uncoated or gelatin-coated PLLA films, cell attachment increased significantly (p < 0.001) on PLLA films coated with ECM gel, fibronectin, or laminin. Myoblasts differentiated into multinucleated myofibers on ECM gel-coated PLLA fibers, and expressed muscle markers such as myosin and alpha-actinin. Oligonucleotide microarray analysis showed similar gene expression profiles for human skeletal muscle cells on ECM gel-coated PLLA fibers as to that observed for myofibers on tissue culture plates. Therefore, PLLA fibers coated with ECM proteins provide a scaffold for the development of skeletal muscle tissue for tissue engineering and cell transplantation applications.

Synergistic improvements in cell and axonal migration across sciatic nerve lesion gaps using bioresorbable filaments and heregulin-beta1.

The success of entubulation for peripheral nerve regeneration is still limited, especially with long lesion gaps. In this study, we examined if regeneration could be enhanced by constructing implants to both align axonal growth and promote Schwann cell proliferation and migration. Silicone implants were used to bridge a 1.4-cm gap in the rat sciatic nerve. Adult female Sprague-Dawley rats were divided into four groups of tubes containing either 1) Matrigel; 2) Matrigel and heregulin; 3) Matrigel and poly(L-lactic acid) (PLLA) microfilaments; or 4) Matrigel, PLLA microfilaments, and heregulin. Ten weeks postimplantation, the number of axons and Schwann cells were measured at the distal end of implants. Implants with microfilaments displayed better tissue cable formation, increased Schwann cell migration, and regeneration of anti-calcitonin gene-related peptide-positive axons, but not RMDO95-positive axons compared with nonfilament-containing groups. Heregulin treatment caused an increase in Schwann cell number, but it demonstrated no significant improvement in either tissue cable formation or axon number. Extensive regeneration was observed through implants containing Matrigel, microfilaments, and heregulin, which induced significant improvements in the number and longitudinal organization of both Schwann cells and axons. These results indicate that physical guidance of microfilaments and the Schwann cell growth factor, heregulin, act synergistically to improve nerve regeneration across long lesion gaps.

Technique paper for wet-spinning poly(L-lactic acid) and poly(DL-lactide-co-glycolide) monofilament fibers.

A simple and repeatable method is described for wet-spinning poly(L-lactic acid) (PLLA) and poly(DL-lactic-co-glycolic acid) (PLGA) monofilament fibers. These fibers are strong, elastic, and suitable for many applications, including use as tissue-engineering scaffolds. The PLLA wet-extruded fibers do not show additional strain-induced crystallization as a result of drawing the fibers during fabrication; however, there is an apparent increase in crystallinity late in the degradation process in saline at 37 degrees C. We have measured the molecular weight degradation in saline at 37 degrees C for fibers of both PLLA and PLGA. Changing solvent systems, polymer blends, and winding rates alters mechanical and morphological properties of these fibers for specific applications. The authors discuss a possible theoretical explanation for these observed changes due to changes in polymer concentration, solvent system, and coagulation bath properties. This wet-extrusion process is simple and inexpensive enough to be carried out in almost any laboratory interested in tissue engineering.

Initial burst measures of release kinetics from fiber matrices.

A comprehensive axisymmetric diffusion model of drug release from a fiber is developed to account for both the initial burst (IB) phenomenon as well as the later diffusion-dominated release. This model is an enhancement over previous models in that a set of four IB parameters are calculated, which both describe the initial burst phenomenon as well as improve the fit for the diffusion-dominated release phase. This model is also an enhancement over previous models in allowing: finite dissolution volumes, finite stirring levels of the medium, and user-specified initial drug dispersion within the device. Five different drug release data sets are used to verify the model and to derive values for the IB parameters. Two of the data sets are from experiments conducted in this study, and the other three sets are from previously published data. These data sets were selected to cover a wide range of possibilities, i.e., from nearly 0% to nearly 100% of the total drug release during IB, yet the model handles all cases equally well.

Expandable bioresorbable endovascular stent. I. Fabrication and properties.

A bioresorbable, expandable poly(L-lactic acid) stent has been designed, based on a linear, continuous coil array principle, by which multiple furled lobes convert to a single lobe upon balloon expansion, without heating. Stent strength and compliance are sufficient to permit deployment by a conventional balloon angioplasty catheter. Several multiple lobe configurations were investigated, with expansion ratios ranging from 1.4 to 1.9 and expanded diameters ranging from 2.3 to 4.7 mm. Compression resistance of the expanded stent is dependent on fiber coil density and fiber ply. A range sufficient for endovascular service was obtained, with less than 4% elastic recoil in six day saline incubation studies. Surface plasma treatment with di(ethylene glycol) vinyl ether significantly reduced platelet adhesion in a 1 h porcine arteriovenous shunt model. Patency was maintained in one week implant studies in the porcine common femoral artery. However, a strong inflammatory response, and significant reduction of the vascular lumen were observed following two weeks implantation. The design principles and fabrication techniques for this bioresorbable stent are sufficiently versatile that a broad range of applications can be addressed. Much work remains to be done, including long-term evaluation of the inflammatory response, and of polymer degradation. The results of this study demonstrate the feasibility of expandable biodegradable stent design and deployment by conventional means.

Bioresorbable polymeric stents: current status and future promise.

Metal stents and, more recently, polymer-coated metal stents are used to stabilize dissections, eliminate vessel recoil, and guide remodeling after balloon angioplasty and other treatments for arterial disease. Bioresorbable polymeric stents are being developed to improve the biocompatibility and the drug reservoir capacity of metal stents, and to offer a transient alternative to the permanent metallic stent implant. Following a brief review of metal stent technology, the emerging class of expandable, bioresorbable polymeric stents is described, with emphasis on developments in the authors' laboratory.

Poly(L-Lactide) microfilaments enhance peripheral nerve regeneration across extended nerve lesions.

After injury, axonal regeneration occurs across short gaps in the peripheral nervous system, but regeneration across larger gaps remains a challenge. To improve regeneration across extended nerve defects, we have fabricated novel microfilaments with the capability for drug release to support cellular migration and guide axonal growth across a lesion. In this study, we examine the nerve repair parameters of non-loaded filaments. To examine the influence of packing density on nerve repair, wet-spun poly(L-Lactide) (PLLA) microfilaments were bundled at densities of 3.75, 7.5, 15, and 30% to bridge a 1.0-cm gap lesion in the rat sciatic nerve. After 10 weeks, nerve cable formation increased significantly in the filament bundled groups when compared to empty-tube controls. At lower packing densities, the number of myelinated axons was more than twice that of controls or the highest packing density. In a consecutive experiment, PLLA bundles with lower filament-packing density were examined for nerve repair across 1.4- and 1.8-cm gaps. After 10 weeks, the number of successful regenerated nerves receiving filaments was more than twice that of controls. In addition, nerve cable areas for control groups were significantly less than those observed for filament groups. Axonal growth across 1.4- and 1.8-cm gaps was more consistent for the filament groups than for controls. These initial results demonstrate that PLLA microfilaments enhance nerve repair and regeneration across large nerve defects, even in the absence of drug release. Ongoing studies are examining nerve regeneration using microfilaments designed to release neurotrophins or cyclic AMP.