RU2758972C9 - Method for making neuron implants - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention relates to medicine, more precisely to neurosurgery and neurology, and is intended for use in neurorehabilitation of patients with neurological diseases of various etiology using implants. To make neutron implants, a tensile layer of insulating material is created, and a tensile conductive pathway is formed on the said layer. Encapsulation of at least one of the above-mentioned conductive paths is carried out by means of an encapsulating layer containing at least one through hole. The hole is filled with an electrically conductive material, conductive paste to predetermined areas of the conductive path is applied. An electrically conductive element is inserted into a ball of electrically conductive paste. The electrical device is filled with a viscous elastomer, and the said elastomer is cured. The tensile layer with conductive paths is made by 3D printing, which includes extrusion of a non-flowing silicone elastomer, polymerization and plasma activation of the surface. The application of electrical channels is carried out by means of inkjet printing using ink in which platinum microparticles are suspended in a viscous solvent. The attachment of flexible metal wires to the contact pads is carried out by the method for point extrusion of the conductive paste, after which the flowing silicone is applied. A suspension of platinum microparticles with a diameter of 0.2-1.8 mcm in triethylene glycol monomethyl ether is used as ink.EFFECT: method allows to expand the range of implants in order to obtain more reliable neuroprostheses, provides stable behavior during stretching in the physiological range of elastic deformation, maintains electrical functionality when exposed to physiologically significant stretches and after long deformation cycles.2 cl, 5 dwg, 3 ex

Description

The invention relates to medicine, more precisely, to neurosurgery and neurology, and can be used for neurorehabilitation of patients with neurological diseases of various etiologies using implants.

Implants are understood as medical devices manufactured to replace lost biological functions, support damaged tissues and expand the capabilities of existing organs. In this case, the surface of implants in contact with biological tissues should consist of a biomedical material in order to better survival in tissues. In some cases, the implants contain electronic devices such as artificial pacemakers and cochlear implants. In particular, cochlear implants can restore hearing in deaf children, deep brain stimulation alleviates symptoms of parkinsonism, and neuromodulation of the spinal cord relieves chronic neuropathic pain.

Currently, neuronal implants are of particular interest, allowing stimulation and recording of the potentials of the central nervous system. This is due to the fact that, according to the World Health Organization, up to 500,000 people in the world annually suffer spinal cord injury. This is often accompanied by severe disorders of sensorimotor and visceral functions, which has significant negative social and economic consequences.

The use of such implants is based on the possibility of artificial control and training of spinal cord neural networks that control sensorimotor functions and can generate locomotor patterns. [Nature Neuroscience 12, 10 (2009), pages 1333-U167].

Most known neuronal implants are matrices of several metal microelectrodes fixed on a biocompatible polymer base (usually polysiloxane, polyimide, or parylene). Such devices, having high flexibility, can be installed on various parts of the nervous tissue, while causing a minimal inflammatory response.

So known neuronal implant (Spinal stimulator systems for restoration of function) [US 9409023], which is a flat array of metal channels enclosed between two layers of biocompatible polymer (parylene). The implant can be used for electrical stimulation for neurorehabilitation of spinal cord injuries. The disadvantages of the implant include the use of layers of parylenes, which have a sufficiently high rigidity (elastic modulus of about 4 GPa) and the use of metal conductors, which leads to sufficient rigidity of the structure, which, in turn, when in contact with mobile tissues (for example, muscle or tissues of the spinal brain) leads to microtrauma, loss of contact or failure of the implant. Therefore, soft neuronal implants made of conductive polymer composites are preferable, the physical and mechanical properties of which are closer to those of body tissues (in particular, in terms of elasticity modulus (Young's modulus), which for spinal cord tissues is, according to various estimates, 0.25-0 .60 MPa.

A neuronal implant for the spinal cord is known [WO 2011157714 A1], made in the form of a plate of 3 layers of material, a contact pad and an electrode, while the first and third layers of the plate are made of non-conductive polysiloxane between which electrically conductive channels are made of a composite of polysiloxane and metal nanoparticles. This device is a multilayer structure in which the electrically conductive channels along with the channels for supplying pharmacological solutions are enclosed between two layers of flexible and elastic polydimethylsiloxane. At certain necessary points on the surface of the insulating layer above the electrically conductive channels, blind holes were made, filled with a composite material polysiloxane - metal nanoparticles; thus creating an electrical contact between the material of the electrically conductive channel and the biological environment. On the opposite side, electrically conductive channels are connected to metal wires through intermediate balls (boluses) of conductive paste for connection to an external electronic device. However, this implant is not without drawbacks, in particular, the use of an intermediate electrically conductive composite material between the surface contact point and the electrically conductive channel reduces the mechanical resistance of the device to cyclic alternating loads that occur during implant operation.

The closest analogue to the claimed invention is a method for manufacturing neuronal implants [US 2018001081], including: providing a stretchable layer of insulating material; the formation on the specified layer of the stretching conductive path; encapsulating said at least one conductive path with an encapsulating layer containing at least one through hole, exposing at least one part of said conductive path and filling said at least one hole with a conductive material, applying a soft and conductive paste or material to predetermined the conductive path region, and inserting the first end portion of the conductive member into a ball of soft conductive paste or material, filling the electrical device with a viscous elastomer, curing said elastomer to polymerize, thereby forming an electrically insulating package. The passivating or encapsulating layer may be a spin-applied and cured silicone layer. The resulting structure is a properly passivated array of conductive paths, eventually formed on a stretchable layer in which the vias are already filled with conductive material. In this case, the conductive material is a mixture of platinum and silicone nanoparticles. The disadvantage of this method is the complex production technology and lack of stability with multiple multidirectional deformations.

The technical task was to expand the range of implants in order to obtain more reliable neuroprostheses. The task was solved by creating a technology that received the code name NeuroPrint.

The technical result was achieved by making a stretchable layer with conductive paths by 3D printing, which includes extrusion of a non-flowing silicone elastomer, polymerization and plasma activation of the surface, deposition of at least one electrical channel by inkjet printing using ink in which platinum microparticles are suspended in a viscous solvent, attaching flexible metal wires to pads by spot extrusion of electrically conductive paste, and applying flowable silicone.

It is optimal to use a suspension of platinum microparticles (diameter 0.2-1.8 μm) in triethylene glycol of monomethyl ether as an ink.

The implant works as follows. The electrode arrays transmit signals both from and to electrogenic tissues via dispersed pads and interconnects embedded in the insulating matrix. For their manufacture, multi-component printing was used using elastic and biocompatible materials. The insulating matrix is first structured by extrusion of a non-flowable silicone elastomer, eg SE 1700; Dow Corning. This determines the overall geometry of the electrode array, as well as the positions of the pad sections (FIGS. 1a(i), (ii)) and interconnect paths (FIGS. 1a(iii)). After polymerization and plasma activation of the surface, the array is ready for the application of electrical channels by means of inkjet printing. The ink used was a suspension of platinum microparticles (diameter 0.2-1.8 µm) in a viscous solvent, for example, triethylene glycol monomethyl ether (TGME), upon subsequent evaporation of which a dense platinum concentrate is formed in the form of the desired electrical conductor (Fig. 1a (iv )). The interfaces for the external electronics are then formed by attaching flexible metal wires to the pads by spot extrusion of an electrically conductive paste (Fig. 1a(v)). In the final step, a flowable silicone such as polydimethylsiloxane (PDMS; SYLGARD 184; DowCorning) is applied to form a conformal impermeable bond with the structure (FIG. 1a(vi)). Due to its low surface tension, PDMS penetrates into the interstices between the particles in the platinum concentrate, forming the composite material in situ.

The inventive method differs from analogues, where the conductor and elastomer are first mixed into a paste and then printed. The advantage of this approach is that the conductive particles in the concentrate form a percolation network prior to PDMS infusion. The mass fraction of platinum in the composite is about 80%. As a result of polymerization of silicone, a composite material with a specific resistance of (9.8±1.5)×10 ^-5 Ohm⋅m (n=4) is obtained, which withstands repeated cycles of tensile deformation.

This approach to hybrid printing results in minimal waste because the conductive concentrate is applied with high precision only where conductivity is required. This facilitates the use of relatively expensive metals such as platinum where biocompatibility requirements limit the range of materials available. Along with platinum, it is possible to use cheaper conductive materials (silver nanowires, carbon nanotubes, etc.) with worse results.

In NeuroPrint's integrated arrays, electrical contact with tissue is facilitated where the platinum particles reach the surface. The developed process of in situ formation of composite materials makes it possible to print electrode arrays of arbitrary planar topology with a mesoscale integration density.

The advantage of the claimed NeuroPrint technology is the use of a set of silicones and composites that ensure that the overall stress-strain behavior of the arrays remains comparable to that of soft tissues such as the dura mater, peripheral nerves or muscles. At the same time, NeuroPrint arrays demonstrate stable behavior during stretching in the physiological range of elastic deformation, retain electrical functionality when exposed to physiologically significant stretching and after long cycles of deformation.

With their simple design, NeuroPrint electrodes eliminate the need for cleanroom processing and the development of special patterns and fabrication tools that are commonly used to manufacture neuronal sensors. Typical production time from design specification to finished prototype can be as little as 24 hours, allowing custom implants to be produced on demand.

The monolithic internal structure, combined with mechanical elasticity, provides resistance to mechanical deformation and efficient transmission of electrical signals from neuronal structures during chronic implantation. The electromechanical properties of the implants made possible their biointegration in long-term in vivo experiments, which indicates their potential application in various fields of translational biomedicine, including neuroprosthetics and brain-computer interfaces.

The industrial applicability of the claimed invention is illustrated by the following examples and drawings.

In FIG. 1 shows the printing scheme and characteristics of planar electrode arrays.

a, Schematic (left) and optical micrographs (right) of each step in the hybrid printing process. (i) The positions of the active centers of the electrodes were determined. The zigzag line in the middle defines the lattice pattern, a design feature that facilitates the diffusion of interstitial fluids through the implant. (ii) Plasma activation is used to improve surface wettability. (iii) Borders are printed to limit the spread of the platinum slurry. (iv) A liquid suspension of platinum microparticles is applied by inkjet printing. To mount the connector, wire guides (silicone; SE 1700) and conductive paste (EPO-TEK H27D, part A) are extruded. (v) The wires for connecting to the electronics are inserted into the guides by hand. (vi) A final low viscosity PDMS seal (SYLGARD 184) is printed. (vii) After thermal polymerization, the finished electrode array is separated from the printed substrate and turned over. Scale mark 1 mm.

b. micrographs from a scanning electron microscope of the contact area (top view) at low (i) and high (ii) magnification. (iii) Cross section of the array illustrating the enclosed interconnect and contact area. The main image shows which parts of the device are shown in the photomicrographs.

c. comparison of the behavior of explants of various soft tissues under tension and deformation with the NeuroPrint array. Data for biological tissues were taken from Noortetal. 20, Kwanetal. 21 and Calvoetal. 22.

d. response to stretching and deformation of a typical NeuroPrint array subjected to cyclic uniaxial deformation. Five successive strain cycles were performed for each of the peak stretch rates of 10% (red), 20% (blue), and 50% (black). For c and d, the length of the test portion of the array was 10 mm and the elongation rate was 0.1 mm/s.

In FIG. 2 shows neuromodulation of locomotor networks using NeuroPrint technology.

a. Use cases in the decerebrated cat model, including spinal cord stimulation (SCS), striated limb and detrusor smooth muscle recording, electrospinogram recording, peripheral nerve stimulation. b. localization of stimulation of the spinal cord and roots (S1-S3; 5 Hz; 0.2 ms). with. Locomotor activity evoked in a decerebrated cat. There is reciprocal activation of the flexor and extensor muscles and coordinated movements of the hind limbs. Mtp, metatarsophalangeal joint; HL, hind limb; L, left; R, right. d-f. Electrical stimulation at the rostrocaudal and mediolateral sites elicited different patterns of hindlimb muscle activation during the gait cycle (n=4 cats, mean ± error of mean). Mean rectified signals from left hip flexor and iliopsoas muscle activity during the stance and swing phase (shaded areas represent 1 s.e.) (d), mean amplitude and area (area under the curve) relative to ipsilateral stimulation (e) show that the electrodes on the left (ipsilateral to the muscle) elicited stronger locomotor impulses during the transfer phase, and that a more rostral stimulation location (f) (above the spinal cord segments and roots S1-S3) increased muscle activity. The significance of e-f was determined by a paired two-tailed t-test.

In FIG. 3 shows activation and monitoring of the neuromuscular system using NeuroPrint technology. a-f, Mixed sciatic nerve stimulation (1 Hz; 0.2 ms), recording ESG and EMG in the medial gastrocnemius muscle provided multilevel access to the sensorimotor system (n=5 cats; mean ± s.e.).

a. wave-like signals generated by a sequential set of fast-conducting proprioceptive, sensory and motor fibers of the sciatic nerve.

b. amplitude of contralateral short-latency ESG evoked potentials relative to ipsilateral potentials evoked by sciatic nerve stimulation.

c. the latency of evoked potentials in ESG depends on the rostro-caudal position of the recording electrode.

d. The latencies observed at various registration points show direct (Ia ESG and motor fiber EMG) and monosynaptic (Ia EMG) responses to sciatic nerve stimulation.

e. Activation of the sciatic nerve reveals the classic suppression of the H-wave by the M-wave.

f. detrusor activity recorded by NeuroPrint electrodes during urodynamic testing (increased detrusor activity can be observed after infusion of saline into the cat's bladder).

g. ESM and EMG recording of two back muscles (rostral and caudal) demonstrate the ability of soft electrodes to access the swimming net in the DanioRerio fish model.

In FIG. 4 shows the biointegration of NeuroPrint electrode arrays.

a. Scheme of a cross section of the spinal cord with a NeuroPrint implant placed in the subdural space, neural network and glial cells in close proximity to the platinum PDMS electrode.

b-f. - Immunohistochemical assessment of neuroinflammation 8 weeks after implantation, where

b. transverse sections of the spinal cord tissue located under the implants.

c. profiles of representative slices of the spinal cord after simulated surgery and NeuroPrint implantation.

d. Normalized optical density of microglia on sections of the spinal cord labeled with gold nanoparticles conjugated with the Iba-1 antibody.

e. representative section fragment containing stained microglia (left) and a graph of the total number of manually counted glial cells per section (right).

f. relative area (left) and optical density (right) of pixels belonging to glial cells.

g-i. - quantitative assessment of movement disorders 8 weeks after implantation, where

g. Schematic representation of walking on a horizontal ladder to assess hindlimb motor control.

h. mean percentage of misses of total steps averaged across all animals and trials (n=8 trials per rat; n=4 rats per group; mean ± s.e.).

i. maximum treadmill speed at which rats could run stably (n=8 attempts per rat; n=4 rats per group; mean ± s.e.).

j-k, side view of a rat on a treadmill. The red dot indicates the position of the metatarsus that was used to analyze the height of the instep (k).

l-n - postural balance and coordination during walking 8 weeks after implantation in implanted and sham-operated rats, where

l. rear view of a rat on a treadmill. Red and blue dots indicate pelvic displacements (m) and lateral foot positions (n), respectively.

about. Paw prints during walking, showing the position of the hind and fore limbs from below, to measure the finer aspects of locomotor control on level ground.

p-q. stride cycle duration (p) and stride length (q) in implanted and sham-operated animals

In FIG. 5 shows the functional stability of NeuroPrint electrode arrays.

a-h. Evaluation of the functional stability of the implant 6-8 weeks after implantation, where

a. Electrode impedance modulus measured at 1 kHz (n=14 total electrodes in four rats), recorded in vitro, immediately after surgical implantation (in intraoperative testing), and then weekly (mean ± s.e.).

b. Dynamics of threshold current (normalized) for evoked potentials in the medial gastrocnemius extensor (n=4 rats; mean values ± s.e.).

c. A typical form of the evoked potential during EMG recording in the medial extensor of the gastrocnemius muscle in response to spinal cord stimulation at 1, 4 and 7 weeks.

d. ESG power spectral density during standing and walking (n=4 rats; mean ± s.e.). HFB, high frequency range; LFB, low frequency range.

e. Thresholds of muscle activation during stimulation (1 Hz; 0.3 ms) of various parts of the spinal cord. The ratio of ipsilateral to contralateral and caudal (S1) to rostral (L3) stimulation is shown (n=4 rats; mean ± s.e.).

f. NeuroPrint implants located above the sensorimotor cortex and in the gastrocnemius muscles of the hind limbs.

g. Left: Electrocorticographic recordings of somatosensory potentials evoked by contralateral (top) and ipsilateral (bottom) stimulation of the gastrocnemius muscle. Signal latency and amplitude are presented in the middle and right panels, respectively (n=5 rats; mean ± s.e.).

h, left: Electromyographic recordings in the medial gastrocnemius (ipsilateral and contralateral) potentials elicited by stimulation of the motor cortex. Center: latency of signals recorded in the ipsilateral and contralateral medial gastrocnemius muscles. Right: Proportion of stimulation pulses that elicited a response (stability) in the medialis gastrocnemius muscles (n=5 rats; mean ± s.e.). a.u., conventional units.

EXAMPLE 1 Obtaining implants is carried out according to the scheme shown in Fig. 1. For their manufacture, multi-component printing was used using elastic and biocompatible materials. The insulating matrix is first structured by extrusion of a non-flowing silicone elastomer, such as, for example, SE 1700; Dow Corning. This determines the overall geometry of the electrode array, as well as the positions of the pad sections (FIGS. 1a(i), (ii)) and interconnect paths (FIGS. 1a(iii)). After polymerization and plasma activation of the surface, the array is ready for the application of electrical channels by means of inkjet printing. When printing, an ink is used in which platinum microparticles (0.2-1.8 µm in diameter) are suspended in triethylene glycol monomethyl ether (TGME), upon subsequent evaporation of which a dense platinum concentrate is formed in the form of the desired electrical conductor (Fig. 1a (iv)) . The interfaces to the external electronics are then formed by attaching flexible metal wires to the pads by spot extrusion of an electrically conductive paste (FIG. 1a(v)). In the final step, a flowable silicone, such as polydimethylsiloxane (PDMS; SYLGARD 184; DowCorning), is applied to form a conformal impermeable bond to the structure. Due to its low surface tension, PDMS penetrates into the interstices between the particles in the platinum concentrate, forming the composite material in situ.

On the basis of this production technology, a library of constructs was created, including the activation of the spinal locomotor system in animals with a motor deficit, the registration of electromyographic activity of striated and smooth muscles for the study of sensorimotor and visceral control. All fabricated designs of electrode arrays have a thickness of about 200 μm.

EXAMPLE 2 The effectiveness of the NeuroPrint electrode was demonstrated using spinal cord electrical stimulation (ESM) in cats. In FIG. Figure 2 shows that continuous EFM (5 Hz; 0.2 ms) of the sacral spinal cord (S1-S3) induced locomotion in decerebrated cats (n=4 cats) with reciprocal activation of flexor and extensor muscles, correct foot position, and well coordinated movements of the hind limbs in the step cycle (Fig. 2a-c). When compared to conventional wire intramuscular electrodes, NeuroPrint demonstrates similar quality of electromyographic (EMG) signals during locomotor activity (Fig. 2c). Moreover, ESM of various rostrocaudal and mediolateral regions triggers local neuronal networks responsible for the specific activation of left-right and rostral-caudal EMG patterns (Figs. 2d-f), which is important for spatiotemporal therapeutic neuromodulation after spinal cord injury.

The next step in testing the capabilities of NeuroPrint was wrapping the NeuroPrint stimulating electrodes around the area of the sciatic nerve of the cat (passing in the proximal part of the thigh), which allowed simultaneous recording of the electrospinogram (ESG) and EMG activity (n=4 cats). Activation of sensory and motor fibers in the sciatic nerve evoked wavelike signals in the spinal cord and gastrocnemius muscle with different latencies (Fig. 3a, d), the shape and amplitude of which depended on the stimulation intensity. We also found that ESG-evoked potentials are modulated by the side of stimulation of the sciatic nerve, as well as by the distance between the recording and stimulation sites (Fig. 3b, c). We also observed the classical dynamics of suppression of the reflex H-wave by the M-wave with increasing amplitude of sciatic nerve stimulation (Fig. 3f). Thus, multilevel monitoring of evoked potentials was carried out using dispersed NeuroPrint electrodes in different parts of the spinal cord.

EXAMPLE 3 Long term biocompatibility and functional stability of NeuroPrint interfaces in a rat model.

Spinal electrode arrays were implanted in the intrathecal space, where repetitive mechanical loads usually place high demands on their stability. The design was supplemented with a lattice pattern as a special feature to improve its integration into the surrounding tissue, ensuring a stable position was maintained during several weeks of testing. Functional stability by behavioral testing and histological sections of implant rats (implant, n=4) and sham-operated (sham, n=4) rats were assessed 8 weeks after surgery (Fig. 5b-i). Sham-operated animals underwent the same surgical procedures (laminectomy, spinal orthosis, connector fixation on the skull) without spinal implants.

Overall, there were no significant differences between the two groups in histological (Fig. 5c-f) and behavioral (Fig. 4g-q) data. As an indicator of neuroinflammation, the expression of the microglial marker Iba1 in sections of the spinal cord was analyzed (Fig. 4d). No significant differences were obtained between the two groups (Fig. 4b-e). In addition to general ability to navigate stairs (Fig. 4g, h) and run on a treadmill at low and high speed (Fig. 4i), fine aspects of sensorimotor activity were monitored, including fore/hind limb coordination (Fig. 4i, j), step cycle duration (Fig. 4p), step width, step length fig. 4q), the height of the instep of the foot (Fig. 54j, k) and their variability. Changes in posture (Fig. 5l-n) of the rats during walking and assessment of mediolateral displacement of the center of mass of the body (Fig. 5l, m) and lateral position of the foot (Fig. 5n) were similar in the Sham group and the implant group. These results confirm the absence of gross and fine motor deficits or imbalances as a result of implantation and indicate a good level of biointegration after 8 weeks.

The functionality of the electrodes was assessed weekly by testing their impedance (Fig. 5a) and current thresholds for muscle recruitment (Fig. 5b, c). Of the 16 electrodes implanted in four rats, 14 retained the initial impedances and the ability to induce evoked muscle potentials. In the first 2 weeks, a trend towards an increase in impedance and current thresholds was observed, which may be associated with the formation of connective tissue (Fig. 6a, b). An assessment was made of the functional state of the neuronal pathways located in the immediate vicinity of the implants (Fig. 4a and 5d-h). At the chronic stage (8 weeks), spinal NeuroPrint interfaces (n=4) were used to record ESG in various behavioral tasks in rats (Fig. 5d), where site-specific neuromodulatory activity of sensorimotor networks was shown (Fig. 4e). Taken together, this indicates a high level of their biointegration and a stable position relative to the target spinal tracts.

Claims (2)

1. The method of obtaining neuronal implants, which includes the creation of a stretchable layer of insulating material, the formation of a stretchable conductive path on the specified layer; encapsulating at least one of the aforementioned conductive paths with an encapsulating layer containing at least one through hole, filling said hole with an electrically conductive material, applying an electrically conductive paste to predetermined areas of the conductive path, inserting an electrically conductive element into a ball of electrically conductive paste, filling the electrical device with a viscous elastomer , the curing of said elastomer, characterized in that the execution of a stretchable layer with conductive paths is carried out by 3D printing, which includes extrusion of a non-flowing silicone elastomer, polymerization and plasma activation of the surface, the application of electrical channels is carried out by inkjet printing using ink, in which microparticles platinum is suspended in a viscous solvent, flexible metal wires are attached to contact pads by spot extrusion of an electrically conductive paste, after which the application of flowable silicone. 2. The method according to claim 1, characterized in that a suspension of platinum microparticles with a diameter of 0.2-1.8 μm in triethylene glycol of monomethyl ether is used as the ink.

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