The field of this invention is medical devices and methods for nerve regeneration.
Several publications are referenced herein. Full citations for these publications are provided below. The disclosures of these publications are incorporated herein by reference in their entirety, unless otherwise noted.
More than 250,000 surgeries are attempted every year to repair damaged nerves. Nerve injuries complicate successful rehabilitation more than any other form of trauma. Painful neuroma formation, often more disabling than its associated sensory deficits, commonly causes major disability. Improvements in the techniques of nerve repair could provide better return of protective sensibility and tactile discrimination, reduce denervation atrophy of muscles, and prevent or minimize pain syndromes.
The nervous system is composed of neurons and glial, or satellite cells. Glial cells include Schwann cells. The neurons carry signals between the brain and the rest of the body, while the Schwann cells provide support for the neurons and enhance the speed of electrical signals. Schwann cells also produce proteins essential for neuron growth (Bunge, 1994; Tortora, 1992). Each neuron has a cell body, an axon, and dendrites. The tip of an axon is the growth cone and is responsible for navigation. Neurons can make multiple contacts with one or more neurons. The organization of the contacts determines the overall function of the nervous system. The axons are surrounded by an insulating layer or myelin sheath formed by the Schwann cells (Tortora, 1992). Injury to the axon that causes the Schwann cells to lose contact with the axons stimulates production of neurotrophic factors such as nerve growth factors. Nerve growth factor (NGF) has been shown to greatly enhance the growth of neurons in culture. With contact, regenerating axons stimulate Schwann cells to proliferate and form a basal lamina of collagen, proteoglycans, and laminin.
When a nerve is severed, a gap is formed between the proximal and distal portions of the injured nerve. In order for the nerve axon to regenerate and reestablish nerve function, it must navigate and bridge the gap. Under the appropriate conditions, e.g., minimal gap length, the proximal end forms neurite growth cones that navigate the gap and enter endoneural tubes on the distal portion. The growth cones sense the extracellular environment and determine the rate and direction of nerve growth. The motion of the axon is responsive to environmental signals provided by other cells that guide the growth cone (Tessier-Lavigne, 1994).
Once the growth cones reach the distal segment, they enter the endoneurial tubes left from the degenerated axons. However, the growth cones and the dendrites on the proximal stump typically grow in many directions and unless the injury is small, the growth cones may never reach the distal segment. The natural ability of the nerve to regenerate is greatly reduced by the disruption of environmental cues resulting from, for example, soft tissue damage, formation of scar tissue, and disruption of the blood supply (Mackinnon and Dellon, 1988; Fawcett and Keynes, 1990, Buettner et al, 1994).
Several techniques have previously been attempted to aid and accelerate the repair of damaged nerves: suturing the severed ends, suturing an allograft or autograft, or regenerating the nerve through a biological or synthetic conduit (Williams et al., 1983; Valentini et al., 1987; Aebischer et al., 1988; Feneley et al., 1991; Calder and Green, 1995).
Autografts and allografts require a segment of a donor nerve acquired from the patient (autograft) or a donor (allograft). The donor nerve segment is removed from another part of the body or a donor and then sutured between the unattached ends at the injury site. Nerve autograft procedures are difficult, expensive, and offer limited success. Often, a second surgical procedure is required and may lead to permanent denervation at the nerve donor site. Allografts typically require the use of immunosuppressive drugs to avoid rejection of donor segments.
Artificial nerve grafts have been used in attempts to avoid the problems associated with autografts and allografts. Artificial grafts do not require nerve tissue from another part of the body or a donor. However, use of artificial nerve grafts has met with only limited success. Axonal regeneration in the peripheral nervous system has only been achieved for graft lengths up to approximately 3 cm in nonhuman primates. There has been little or no success with longer grafts. The previously used artificial nerve grafts were unsuitable for bridging longer gaps between distal and proximal nerve stumps and, therefore, are unsuitable for treating a significant proportion of nerve injuries.
Neurite growth has been aided to a limited extent by fabricating grooves on substrate surfaces (Weiss, 1945; Turner, 1983; Clark et al., 1987; Dow et al., 1987). The grooves employed in these studies were engraved on plastic or quartz substrates. The substrates utilized are unsuitable for implantation in vivo and thus the authors were unable to determine if the grooves could guide neurite growth in an animal. Alignment of neurons using physical guidance cues alone is highly uncertain and difficult to reproduce. For example, the neurites are typically aligned on only part of the substrate and unaligned on other parts and exhibit undesireable axon branching.
Tai et al., 1998 refer to the effects of micropatterned laminin glass surfaces on neurite outgrowth and growth cone morphology. In Tai et al., micropatterns consisting of laminin stripes alternating with glass stripes were formed on glass coverslips. Neuronal cultures were prepared from chicken dorsal root ganglia and seeded on either micropatterned laminin coverslips or on a uniform laminin coated glass surface. While neuronal growth on the micropatterned laminin surface was biased in the direction of the pattern, severe axon branching formed dense axon outgrowth. Thus, while the laminin provided some level of chemical guidance, applicability of the technique was limited. In addition, the glass substrates are unsuitable for implantation into patients.
Biodegradable conduits filled with magnetically aligned collagen rods have also been used in an attempt to provide directional guidance to regenerating neurons. However, this approach does not provide any chemical guidance to regenerating neurons and has had only limited success. The presence of the collagen rods reduces the space available for neuronal outgrowth, constricts growth, does not reduce axonal branching, and limits the natural transport of oxygen, nutrients, and waste products.
Preferred embodiments of the present invention provide methods and apparatus for regenerating nerves utilizing substrates having a surface containing grooves, as described herein, and chemical, cellular and/or electrical cues (collectively and individually referred to as xe2x80x9cguidance factorsxe2x80x9d) provided in the grooves to obtain the desired nerve growth rates and to regain nerve functionality. Especially preferred nerve growth guidance factors include Schwann cells, stem cells and laminin. The combination of the substrates and guidance factors and methods according to the invention results in accelerated neurite elongation rates, excellent neurite alignment along the substrate grooves, and restored nerve functionality.
In a preferred embodiment of the invention, methods and associated apparatus for regenerating severed nerves are provided comprising a substrate having a surface containing one or more substantially linear grooves, wherein said one or more grooves contain one or more guidance factors for nerve regeneration. The substrate is preferably positioned at an end of a severed nerve such that the grooves are substantially coextensive to the severed nerve end and the nerve is allowed to grow into one or more grooves of the substrate. The grooves preferably contain one or more guidance factors for nerve regeneration.
In another preferred embodiment of the invention, the substrate is in the form of a cylinder and the grooves are disposed on the surface of the inner wall of the cylinder. In a further preferred embodiment, the guidance conduit is porous. The conduit is preferably implanted into an animal and sutured to the ends of a severed nerve to achieve directional nerve growth and regeneration.
In particularly preferred embodiments of the invention the substrate is formed from poly(D,L-lactide) or copolymers of lactic and glycolic acids. In a further preferred embodiment, the substrate also comprises nerve growth inhibitors or xe2x80x9cnegative guidance factorsxe2x80x9d (e.g., poly(vinyl alcohol)) to direct and limit neuronal growth to the grooves on the surface of the substrate. According to this preferred embodiment of the invention, one or more negative guidance factors are disposed between the grooves on surface of the substrate. The negative guidance factors inhibit neuron growth outside of the grooves and prevent axon branching of the neuronal outgrowth.
In yet another preferred embodiment, at least one electrode is positioned within said one or more grooves. The combination of preferred guidance factors (e.g., Schwann cells, laminin, and stem cells) and electrical signals generated by the electrode provide further stimulation to orient nerve growth along the axis of the grooves.
The above and other characteristics and advantages of the invention can be better understood from an analysis of the following written description and the accompanying drawings, where like reference numbers represent like elements, or may be learned by practice of the invention.
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