Biomimetic implants

Abstract

The present invention provides implants for spinal cord and peripheral nerve injuries and methods for treating neuronal injuries using the implants. [Solution] The implant may include a three-dimensional printed structure including a first end and a second end, one or more channels beginning at the first end and ending at the second end, and at least one type of stem cell contained in at least one channel.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 62 / 433,142, filed December 12, 2016, the disclosure of which is incorporated herein by reference in its entirety.

[0002] (Statement regarding federally sponsored research and development) This invention was made with government support under Grant No. EB014986 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] (Technical field) Disclosed herein are three-dimensional biomimetic implants containing stem cells for the treatment of spinal cord and nerve injuries thereof. [Background technology]

[0004] Methods for bioprinting functional tissues face many challenges, most notably the lack of suitable biofabrication techniques for constructing the complex three-dimensional (3D) microarchitectures essential for guiding cell proliferation and promoting tissue maturation. 3D printing of central nervous system structures has been unsuccessful to date. Summary of the Invention [Means for solving the problem]

[0005] Described herein are implantable devices or implants for tissue repair. In some embodiments, the tissue can be spinal cord tissue or peripheral nerve tissue. These implants can be used to treat injuries to either of these types of tissue.

[0006] The implant can include a layerless three-dimensionally printed structure, hi some embodiments, the implant can include a three-dimensionally printed structure including a first end and a second end, one or more channels beginning at the first end and terminating at the second end, and at least one type of stem cell contained in at least one channel.

[0007] In some embodiments, the implant is a biomimetic. The implant may be a biomimetic for the spinal cord, comprising a core (representing the spinal cord gray matter) and a shell (representing the spinal cord white matter), with the shell comprising channels. In other embodiments, the implant may be a biomimetic for a peripheral nerve, comprising a honeycomb structure of linear channels packed together. In the case of the spinal cord or peripheral nerve, the linear channels may guide regenerating axons to different sides of the lesion.

[0008] Thus, disclosed herein is a biomimetic implant for spinal cord or peripheral nerve injury, the implant comprising a three-dimensional (3D) implant including a first end and a second end, a core and a shell, mimicking the structure of the injury site, at least one channel in the shell beginning at the first end and ending at the second end, and at least one type of stem cell contained in the at least one channel.

[0009] In some embodiments, the implant is manufactured by 3D printing.

[0010] In some embodiments, at least one kind of stem cell is a neural stem cell. In some embodiments, the neural stem cell is an embryonic stem cell, an iPSC-derived stem cell, a directly differentiated neural stem cell, or a combination thereof. In some embodiments, at least one kind of stem cell is a mesenchymal stem cell. In some embodiments, the stem cell is engineered to express BDNF, NT3, GDNF, or a combination thereof.

[0011] In some embodiments, the three-dimensional printed implant comprises polyethylene glycol diacrylate or gelatin methacrylol, or a combination thereof.

[0012] In some embodiments, the implant is a biomimetic for the spinal cord. In some embodiments, the implant is a biomimetic for a peripheral nerve.

[0013] In some embodiments, the channels are linear. In some embodiments, the channels are parallel to one another. In some embodiments, the channels guide regenerating axons from a first end to a second end. In some embodiments, the implant comprises two or more channels with hexagonal cross sections clustered as a honeycomb structure.

[0014] Also disclosed herein is a method for treating nerve injury in a host in need thereof, the method comprising implanting a biomimetic implant as disclosed herein at a location in need of treatment and allowing cells to regenerate at the site of injury.

[0015] In some embodiments, the nerve injury is a spinal cord injury, a motor-complete spinal cord injury, a motor-incomplete spinal cord injury, or a peripheral nerve injury. In some embodiments, the nerve injury is a spinal cord injury. In some embodiments, the nerve injury is a peripheral nerve injury.

[0016] In some embodiments, the method further comprises administering physical therapy to the recipient.

[0017] Also disclosed herein is a method of manufacturing the biomimetic implants disclosed herein, the method comprising scanning the location of the spinal cord or peripheral nerve of a recipient requiring treatment to determine the area of damage, and three-dimensionally printing the implant to surround the damaged area. [Brief explanation of the drawings]

[0018] [Figure 1A] Figure 1A shows the 3D-printed implant mimicking the spinal cord structure. The 3D printer setup includes a UV light source (365 nm wavelength), a computer for slice image flow generation and system synchronization, a digital micromirror device (DMD) for optical pattern generation, a set of projection optics, a stage for sample position control, and a CCD imaging system for online monitoring of the fabrication process. [Figure 1B] Figure 1B shows a 3D printed implant that mimics the spinal cord structure. Figure 1B illustrates microscale continuous projection 3D printing (μCPP) layerless 3D printing, which forms a structure without discrete layers, as is often seen with inkjet 3D printers. [Figure 1C] Figure 1C shows a 3D-printed implant that mimics spinal cord structure. Figure 1C shows heavy chain neurofilament (NF200) labeling of axons in an intact T3 rat spinal cord. The left image shows the rostral portion, and the right shows the caudal portion. Axons in the white matter (top of panel) are highly organized in parallel arrays moving from rostral to caudal, while axons in the gray matter (bottom of panel) do not exist in linear arrays. The disclosed implant mimics the linear organization of white matter. The linea alba defines the boundary between white and gray matter. [Figure 1D] Figure 1D shows a 3D-printed implant mimicking the spinal cord structure. Figure 1D illustrates the projections of different axonal tracts (bundles) in the dorsolateral quadrant of a T3 rat spinal cord. The rubro-rubrospinal tract (Ru), raphe-anterior raphe-spinal tract (Ra), reticulo-olivo-spinal tract (Ret), proprio-propriospinal tract (Pr), spinothalamic-spinal tract (ST), and CST-corticospinal tract (CST) are shown. The butterfly-shaped area in the center represents the implant's core (resembling the "gray matter" of a normal spinal cord), while the remainder of the figure represents the implant's shell (resembling the "white matter" of a normal spinal cord). [Figure 1E]Figure 1E shows a 3D-printed implant that mimics spinal cord structure. The figure shows the guidance achieved in the rostro-caudal axis, thereby guiding regenerating axons to their appropriate tracts on the far side of the lesion. The arrows pointing to the entry and exit points of regenerating axons within the implant demonstrate that the implant maintains precise 3D coordinates throughout the lesion site, matching the natural host architecture. [Figure 2] 1 shows mechanical measurements of the elastic modulus of the implants using dynamic mechanical analysis (DMA). [Figure 3A] Figure 3A shows an exemplary spinal implant disclosed herein. Figure 3A shows a mid-sagittal cervical T1-weighted magnetic resonance (MR) image of a clinically intact human (ASIA A) spinal cord injury. A strip of damaged host white matter is evident anterior (to the right) of the lesion (arrow). [Figure 3B] 3A and 3B show an exemplary spinal implant as disclosed herein. FIG. 3B shows a traced outline of the cystic lesion cavity from FIG. [Figure 3C] 3A-3C show an exemplary spinal implant as disclosed herein. FIG. 3C shows a computer-aided design (CAD) 3D model of the 3D printed implant corresponding to the exact lesion shape. [Figure 3D] 3A-3D show an exemplary spinal implant as disclosed herein. FIG. 3D shows the printed implant. [Figure 3E] 3A-3D illustrate an exemplary spinal implant as disclosed herein. FIG. 3E illustrates a hypothetical fit of the printed 3D implant of FIG. 3D into a human contusion cavity. [Figure 4] Figure 4 shows a 3D-printed implant implanted into a spinal cord injury site 4 weeks after implantation. Figure 4 shows a cross-sectional image of the implant at the lesion site labeled for axons (neurofilament NF200) (cross section), demonstrating that the overall implant structure remains intact 4 weeks after implantation. The scale bar is 500 μm. (core). [Figure 5A]Figure 5A shows a 3D-printed implant implanted into a spinal cord injury site 4 weeks after implantation. Nissl staining of the implantation site (site of a T3 complete transection) reveals a reactive cell layer (arrow) at the site of implantation of the agarose scaffold (Figure 5A). Scale bar: 200 μm. Left: rostral; right: caudal. Interrupted line defines the boundary between the recipient spinal cord and the implant. (Scaffold). [Figure 5B] Figure 5B shows a 3D-printed implant implanted at the site of spinal cord injury 4 weeks after implantation. The 3D-printed polyethylene glycol diacrylate / gelatin methacrylol (PEGDA / GelMa) implant disclosed herein is substantially attenuated after implantation (Figure 5B). Scale bar is 200 μm. Left: rostral; right: caudal. Interrupted line defines the boundary between the recipient spinal cord and the implant. (Scaffold) [Figure 5C] 3D printed implants implanted into the spinal cord injury site 4 weeks after implantation. Quantification of reactive cell layer (RCL) thickness ± SEM is shown. *p<0.05 (Student's t-test). [Figure 6A] Figure 6A shows a 3D-printed implant implanted at the site of spinal cord injury 4 weeks after implantation. The recipient glial scar (Figure 6A) is revealed by glial fibrillary acidic protein (GFAP) immunoreactivity in an animal with lesion only (no implant). Scale bars in Figure 6A are 250 μm and 100 μm in Figure 6C. A GFAP-labeled barrier is absent around the 3D-printed PEGDA / GelMa implant; instead, glial fibers are arranged longitudinally in grooves where they could potentially support outgrowth axons. (Lesion: Injury) [Figure 6B]Figure 6B shows a 3D-printed implant implanted into the spinal cord injury site 4 weeks after implantation. The agarose scaffold in the lesion area is shown. The scale bars in Figure 6B are 250 μm and 100 μm, respectively, in Figure 6C. GFAP-labeled barriers are absent around the 3D-printed PEGDA / GelMa implant; instead, glial fibers are arranged longitudinally in grooves where they could potentially support outgrowth axons. (Agarose: Agarose) [Figure 6C] Figure 6C shows the 3D-printed implants implanted into the spinal cord injury site 4 weeks after implantation (rostral to the left, caudal to the right). GFAP-labeled barriers are absent around the 3D-printed PEGDA / GelMa implants; instead, glial fibers are arranged longitudinally in grooves where they could potentially support outgrowth axons. [Figure 6D] 3D printed implants implanted into the spinal cord injury site 4 weeks after implantation. Quantification of GFAP intensity in the recipient spinal cord surrounding the injury site ± SEM is shown. *p<0.05 (ANOVA with post-hoc Tukey's test). [Figure 7A] Figure 7A shows a 3D-printed implant implanted into the spinal cord injury site 4 weeks after implantation. The implant is well vascularized (RECA-1 immunolabeling for blood vessels) (Figure 7A). The scale bar in Figure 7A is 25 µm. [Figure 7B] Figure 7B shows a 3D-printed implant implanted into the spinal cord injury site 4 weeks after implantation. Toluidine blue staining indicates blood vessels (asterisks) (Figure 7B). The scale bar in Figure 7B is 20 µm. [Figure 8A] Figure 8A shows a 3D-printed implant implanted into the spinal cord injury site 4 weeks after implantation. NF200-labeled recipient axons are unable to cross the scar surrounding the agarose scaffold (Figure 8A). Scale bar: 100 μm. The dashed line indicates the entrance point of the implant from the rostral aspect of the lesion site. (Agarose). [Figure 8B]Figure 8B shows a 3D-printed implant implanted into the spinal cord injury site 4 weeks after implantation. NF200-labeled recipient axons readily penetrate the 3D-printed implant (Figure 8B). The scale bar is 100 μm. The dashed line indicates the entrance point of the implant from the rostral aspect of the lesion site. [Figure 9A] Figure 9A shows a 3D-printed implant implanted at the site of spinal cord injury 4 weeks after implantation. Figure 9B is an electron micrograph image within the channel showing an axon (asterisk) associated with an adjacent ensheathing Schwann cell (Sc). The scale bar is 1 μm. [Figure 9B] Figure 9B shows a 3D-printed implant implanted at the site of spinal cord injury 4 weeks after implantation. Figure 9B shows a magnified view of the channel from Figure 9A, showing S100-labeled Schwann cells (arrows) surrounding NF200-labeled axons. The scale bar is 5 μm. [Figure 9C] Figure 9C shows a 3D-printed implant implanted into a spinal cord injury site 4 weeks after implantation. Figure 9D is an electron micrograph of a channel showing myelinated axons in the implant with Schwann cells (SCs). The scale bar is 0.5 μm. [Figure 10A] Figure 10 shows a 3D-printed implant disclosed herein loaded with neural stem cells four weeks after implantation in a rat. Figure 10A shows a channel (horizontal section) filled with GFP-expressing neural stem cells (arrows). Scale bar is 200 μm. (Fill: filling material, Wall: wall). [Figure 10B] Figure 10B shows a 3D-printed implant disclosed herein loaded with neural stem cells four weeks after implantation in a rat. Figure 10B shows the rostral entrance to the channel penetrated by recipient NF200-labeled axons, which are distinguished from graft-derived axons by the absence of GFP expression. Scale bar 50 μm. [Figure 10C] Figure 10 shows a 3D printed implant disclosed herein incorporating neural stem cells four weeks after implantation into a rat. Figure 10C shows that the implanted neural stem cells extend GFP-expressing axons that are linearized by the implanted linear architecture. [Figure 10D] Figure 10D shows a 3D-printed implant disclosed herein loaded with neural stem cells four weeks after implantation in a rat. 5HT-labeled recipient serotonergic axons enter the stem cell-filled channel from the rostral end (left) of the lesion and regenerate linearly within the channel (arrow). Scale bar is 100 μm. [Figure 10E] Figure 10 shows a 3D-printed implant disclosed herein incorporating neural stem cells four weeks after implantation in a rat. Figure 10A shows a channel (horizontal section) filled with GFP-expressing neural stem cells (arrows). Figure 10E shows serotonergic axons regenerating linearly into an empty implant lacking stem cells, although the number of axons penetrating is reduced. Scale bar is 100 μm. [Figure 10F] Figure 10 shows a 3D-printed implant disclosed herein incorporating neural stem cells four weeks after implantation in a rat. Figure 10F shows 5HT-labeled recipient serotonergic axons regenerating into the caudal end of the stem cell-containing implant and respecting the linear boundary created by the implant structure. Scale bar is 50 μm. [Figure 10G] Figure 10G shows 3D printed implants disclosed herein incorporating neural stem cells four weeks after implantation in rats. Quantification of 5HT axons reaching the tail of the implant is shown. *p<0.05 (ANOVA, +SEM). [Figure 10H] Figure 10H shows a 3D-printed implant disclosed herein incorporating neural stem cells four weeks after implantation into a rat. 5HT-labeled motor axons exit the tail of the channel (arrow) to regenerate into the recipient spinal cord distal to the lesion. This line defines the exit point from the tail channel into the caudal spinal cord. Scale bar: 50 μm. (Host Caudal: recipient caudal), (Spinal Cord: spinal cord). [Figure 11] At the ultrastructural level, axons of various diameters are present within the channels, and many axons are myelinated after implantation of the implant in Figure 10. Scale bar: 500 μm. [Figure 12A] Ultrastructural analysis of the implantation site 4 weeks after implantation. Figure 12A shows that axons of various diameters (asterisks) are present within the channel, and many axons are myelinated (M). Scale bar is 500 nm. [Figure 12B] Figure 12B shows an ultrastructural analysis of the transplant site 4 weeks after transplantation. Figure 12B shows oligodendrocytes sending out multiple processes to myelinate and ensheath the axons. Scale bar is 0.2 μm. [Figure 13] Shown is a printed implant loaded with neural stem cells 4 weeks after implantation. Synapses (arrows) form between axons in the channel and dendrites of the transplanted neural stem cells. Scale bar is 200 μm. The synapses are asymmetric, and the presynaptic boutons contain round vesicles, indicating that they are excitatory. [Figure 14A] Figure 14A shows a printed implant loaded with neural stem cells 4 weeks after transplantation. Figure 14A shows 5HT recipient axons regenerating into the transplant channel forming appositional contacts (arrows) with dendrites (labeled with Map2) of transplanted neural stem cells (GFP) 4 weeks after transplantation. Scale bar is 10 μm. [Figure 14B] Figure 14B shows printed implants incorporating neural stem cells 4 weeks after implantation. Figure 14B shows quantification of 5HT axons reaching the tail of the implant. *p<0.05 (ANOVA p<0.01, post-hoc Tukey's test P<0.01 comparing both NSC implant groups with the NSC graft-only group and the empty implant group). [Figure 15A] Figure 15A shows a printed implant loaded with neural stem cells in a long-term in vivo study. Figure 15A shows the anatomy 6 months after implantation. Figure 15A: The channel remains structurally intact and is filled with GFP-expressing neural stem cells. Horizontal section, rostral to the left. (Fill: filling, Wall: wall). [Figure 15B]Figure 15B shows the printed implant incorporating neural stem cells in a long-term in vivo study. Figure 15B shows the anatomical structure 6 months after implantation. Figure 15B: Corticospinal axons labeled anterogradely with RFP enter the implant and extend linearly caudally, aligned by the implant structure (Rostral Entrance). [Figure 15C] Figure 15C shows a printed implant incorporating neural stem cells in a long-term in vivo study. The anatomy is shown 6 months after implantation. Horizontal section 15C: Corticospinal axon (CST) axons converge within the channel onto NeuN-labeled neurons, forming potential bouton-like contacts with the soma. [Figure 15D] Figure 15D shows the printed implant incorporating neural stem cells in a long-term in vivo study. The anatomy is shown 6 months after implantation. Figure 15D: GFP-immunoreactive axons extend from the implant into the white and gray matter recipients caudal to the lesion. Ventrolateral white matter, 2 mm caudal to the lesion. [Figure 15E] Figure 15E shows a printed implant incorporating neural stem cells in a long-term in vivo study. The anatomy is shown 6 months after implantation. Figure 15E: GFP-labeled axons derived from neural stem cells (NSCs) form potential bouton-like structures on gray matter NeuN-immunoreactive recipient neurons located 2 mm caudal to the lesion. [Figure 15F] Figure 15F shows printed implants incorporating neural stem cells in a long-term in vivo study. Figure 15F shows behavioral studies. Figure 15F: Neural stem cell / implant-treated animals showed significant functional recovery on the BBB locomotor scale 5 months after implantation, reflected by consistent movement of each of the three joints in both hind limbs (**p<0.05, *p<0.01). [Figure 15G]Figure 15G shows a printed implant incorporating neural stem cells in a long-term in vivo study. Behavioral studies are shown in Figure 15G. Figure 15G: Schematic of an electrophysiological study performed 6 months after implantation. Transcranial electrical stimulation was applied to the motor cortex in the brain, and motor evoked potentials (MEPs) were recorded from the hind limbs. [Figure 16A] Figure 16A shows printed implants loaded with neural stem cells 6 months after implantation. Rats receiving 3D printed stem cell implants show partial recovery of MEP responses (Figure 16A). [Figure 16B] Figure 16B shows a printed implant loaded with neural stem cells 6 months after implantation, with this recovery being abolished by subsequent resection of the umbilical cord over the implant. [Figure 16C] Printed implants loaded with neural stem cells are shown 6 months after implantation, with no MEP recovery observed in animals with empty implants (Figure 16C). [Figure 16D] Figure 1 shows printed implants incorporating neural stem cells 6 months after implantation, demonstrating that mean MEP amplitudes are significantly greater in animals implanted with neural stem cell-containing implants (p<0.01). [Figure 17A] Longitudinal images of 2 mm printed implants of different lengths (FIG. 17A) are shown. The scale bar is 0.5 mm. [Figure 17B] Longitudinal images of 4 mm printed implants of different lengths (FIG. 17B) are shown. Scale bar is 0.5 mm. [Figure 18A] Nissl staining of agarose (Figure 18A) shows persistence of implant structure in agarose and PEGDA after 4 weeks, and degradation of the hyaluronic acid scaffold. Scale bar is 250 μm. (Agarose) [Figure 18B]Nissl staining of PEGDA / GelMa (Figure 18B) shows persistence of implant structure in agarose and PEGDA after 4 weeks, and degradation of the hyaluronic acid scaffold. Scale bar is 250 μm. (Gelatin) [Figure 18C] Nissl staining of the hyaluronic acid implant (Figure 18C) shows persistence of implant structure in agarose and PEGDA after 4 weeks, and degradation of the hyaluronic acid scaffold. Scale bar is 250 μm. (Hyaluronan) [Figure 18D] Figure 1 shows that RCL thickness is significantly reduced in PEGDA-GelMa implants (p<0.05, ANOVA; post-hoc Tukey's test comparing PEGDA group to agarose and HA scaffolds). Mean ± s.e.m. [Figure 19] Implant degradation measured by a decrease in wall thickness is shown. *p<0.0001, **p<0.001. (ANOVA; post-hoc Tukey's test). [Figure 20A] Figure 20A shows neuronal regeneration 4 weeks after implantation. Figure 20A shows GFP-labeled implants from four different animals, demonstrating complete and uniform filling of the channels with rodent neural stem cells, which also occupy the interface between the implant and the recipient (arrows). Scale bar = 0.5 mm. [Figure 20B] Neuronal regeneration 4 weeks after transplantation is shown. [Figure 20C] Neuronal regeneration 4 weeks after transplantation is shown. [Figure 20D] Neuronal regeneration 4 weeks after transplantation is shown. [Figure 21A] In addition to GFP, stem cell-derived cells in the channel express the neuronal marker Hu (FIG. 21A). Scale bar is 5 μm. [Figure 21B] In addition to GFP, stem cell-derived cells in the channel express NeuN (FIG. 21B). Scale bar is 5 μm. [Figure 21C]They express GFP as well as the oligodendrocyte marker Olig2. Scale bar: 5 μm. [Figure 21D] The astrocyte marker GFAP is shown together with GFP. Scale bar is 5 μm. [Figure 22] 1 shows the distribution of stem cell differentiation markers in grafted cells within the channel. [Figure 23] Serotonergic axons (arrows) are visible in stem cell grafts injected into the lesion site without an implant; the axons are vertically oriented and therefore misaligned with the rostral-caudal axis of the lesion site. The interrupted line defines the graft-host interface. Left: rostral; right: caudal (lesion site). Scale bar: 50 μm. [Figure 24A] Stem cells do not fill the channels (Figure 24A), and the tail of the implant contains substantially fewer 5HT-labeled axons. Scale bar, 50 μm. [Figure 24B] Stem cell grafts without stem cells implanted (Figure 24B) contain substantially fewer 5HT-labeled axons at the tail of the implant. Scale bar 50 μm. [Figure 25] 3D rendering of a 10 μm z-stack of 5HT-labeled recipient motor axons in a channel that was not co-labeled with GFP, demonstrating the absence of serotonergic neuronal cell bodies in spinal cord-derived neural stem cell grafts. [Figure 26A] (FIG. 26A, scale bar 100 μm) 5HT-labeled motor axons are seen in the recipient spinal cord caudal to the lesion in the recipient gray matter. NF200 staining for recipient axons is seen. [Figure 26B] (FIG. 26B, scale bar 50 μm) 5HT-labeled motor axons are seen in the recipient spinal cord caudal to the lesion in the recipient white matter. NF200 staining for recipient axons is seen. [Figure 26C](Figure 26C, scale bar 25 μm) 5HT-labeled motor axons are seen in the recipient spinal cord caudal to the lesion at the crossover from white to gray matter. NF200 staining for recipient axons is seen. [Figure 27A] A single channel of a 3D-printed implant loaded with neural stem cells showing vascularization. GAP43-labeled axons inside a channel loaded with GFP-expressing NSCs (FIG. 27A, scale bar is 25 μm). [Figure 27B] A single channel of a 3D-printed implant loaded with neural stem cells is shown, demonstrating vascularization. GAP43-labeled axons in the recipient spinal cord caudal to the implant (FIG. 27B, scale bar is 25 μm). [Figure 28A] Toluidine blue staining is shown, with arrows pointing to blood vessels (FIG. 28A, scale bar is 100 μm). [Figure 28B] Toluidine blue staining is shown, with arrows pointing to blood vessels, which are marked with asterisks in the EM image (Figure 28B, scale bar 1 μm). [Figure 29] Figure 1 shows that PDGFR labeling of pericytes revealed surrounding RECA-1 labeled blood vessels, indicating BBB restoration. Scale bar is 15 μm. [Figure 30] MEP latencies were shorter in animals implanted with stem cell implants compared to empty implant-treated animals, and were close to those observed in intact animals (p<0.01). [Figure 31] 1 shows motor evoked potential recordings from animals receiving implants with or without stem cells. [Figure 32A] 1 shows an implant containing a linear channel. [Figure 32B] FIG. 32B is an enlarged view of FIG. 32A. [Figure 33A] 1 shows an implant comprising a honeycomb structure of inter-packed linear channels. [Figure 33B] FIG. 33B is an enlarged view of FIG. 33A. DETAILED DESCRIPTION OF THE INVENTION

[0019] Disclosed herein are implants and methods for rapid three-dimensional (3D) microscale printing of regeneration-promoting implants that biomimetic the complex, fascicular microscale structure of the spinal cord or peripheral nerves. The polymer-based implants can be rapidly printed and scaled to fit clinically relevant spinal cord or peripheral nerve sizes and lesion geometries. Injured host axons regenerate into the 3D biomimetic implant and synapse with neural stem cells implanted in the device. The implanted neural stem cells then extend axons from the implant into the host spinal cord or peripheral nerve beneath the injury, restoring synaptic transmission and significantly improving functional outcomes. A new, alternative electrophysiological relay across the injury site forms, supporting significant functional and motor improvements. Thus, complex 3D biomimetic implants provide a means to promote central nervous system regeneration through precision medicine.

[0020] Described herein are medical implants that aid in the restoration of bodily function. Function can be restored in mammals, which can include humans, horses, pigs, cows, bulls, goats, sheep, dolphins, dogs, cats, camels, and the like. In one embodiment, the mammal is a human. In some embodiments herein, the mammal is referred to as a recipient.

[0021] These medical implants may be used in some embodiments to promote axonal regeneration after spinal cord injury or peripheral nerve injury.

[0022] In some embodiments, the medical implant comprises a three-dimensional implant and optionally stem cells. In some embodiments, the implant is manufactured by 3D printing. These implants can be custom designed to fit a specific patient's anatomy. The terms "scaffold" and "implant" are used interchangeably and refer to 3D printed structures with or without stem cells.

[0023] Bioengineered scaffolds or implants support axonal regeneration at spinal cord or peripheral nerve lesion sites, but these technologies have been limited by foreign body reactions at the implantation site, cumbersome manufacturing requirements, limitations in scalability to human-scale injuries, and a lack of biomimicry of the native spinal cord or peripheral nerve. The implants and methods using implants described herein include structures that biomimetic the complex fascicular structure of the spinal cord or peripheral nerve. Printable implants can be easily and quickly manufactured, reduce foreign body reactions, and / or support linearly aligned host axonal regeneration across the lesion site. Furthermore, neural stem cells can be incorporated into the implant. Stem cells can support regenerating host axons as they bridge across and beyond the lesion site, promoting functional regeneration in vivo.

[0024] The spinal cord is used as a template for designing spinal implants (Figure 1A). Microchannels are included to provide alignment of the implant channel with the host axon tract above and below the injury (Figure 1D-E). Because the inner "gray matter" region of the spinal cord typically does not contain axons projecting below the injury site, this component of the implant, the core, is designed as a solid region that enhances the structural integrity of the implant (Figure 1D). The use of agarose microchannel implants has shown that 80% of host axons entering the lesion site can be guided by linear or parallel conduits to reach the opposite (caudal) end of the lesion. However, agarose induced a foreign body response consisting of a collagen-based reactive cell layer that attenuated and trapped axons within the implant, preventing axonal growth beyond the channel. Thus, disclosed herein is an implant fabricated from a mixture of degradable materials that reduces the reactive cell layer due to a reduced foreign body response, allowing host axons to better pass through and even cross the lesion.

[0025] Materials used to form the implant include biologically acceptable polymers. In some embodiments, the polymer may include polyethylene glycol-based polymers, such as, but not limited to, polyethylene glycol diacrylate (PEGDA) and poly(ethylene glycol) diacrylamide. In some embodiments, the polymer may include gelatin methacrylol (GelMA) hydrogel. In some embodiments, the polymer may include a combination of polyethylene glycol diacrylate, poly(ethylene glycol) diacrylamide, and gelatin methacrylol. In some embodiments, the polymer may include a combination of polyethylene glycol diacrylate and gelatin methacrylol. In some embodiments, the polymer may include a combination of poly(ethylene glycol) diacrylamide and gelatin methacrylol.

[0026] In some embodiments, the biocompatible material PEGDA is used as the implant material. PEGDA itself is non-adhesive to cells; therefore, gelatin methacrylate (GelMa), a photopolymerizable denatured collagen that retains cell-binding ligands and matrix metalloproteinase degradation sites, is included to support cell attachment to the implant wall and long-term cell viability. Because a mismatch in mechanical properties between the implant and the host can lead to compression or tearing at the spinal cord or peripheral nerve interface, causing integration failure, various concentrations of each material and crosslink densities of the printed implants were tested until a combination was identified that mimicked the mechanical properties of native spinal cord or peripheral nerve tissue.

[0027] An advantage of 3D bioprinting is the ability to rapidly print implants of different sizes and irregular shapes to fit individual patient lesion sites, which can be identified by magnetic resonance imaging (MRI). As shown in Figure 3A-E, implants formed from PEGDA / GelMa were printed to fit the exact shape of a human spinal cord lesion cavity according to MRI. Implants that fit the morphology of even more complex human injury cavities have been printed.

[0028] The implants disclosed herein comprise a core and a shell, the core resembling the "gray matter" portion of a normal spinal cord or peripheral nerve, and the shell resembling the "white matter" portion of a normal spinal cord or peripheral nerve.

[0029] The implant may include one or more channels. In some embodiments, the channel is in the shell. In some embodiments, the one or more channels extend from a first surface to a second surface. In some embodiments, the first surface is an upper surface and the second surface is an lower surface. In some embodiments, the first surface is an lower surface and the second surface is an upper surface. In some embodiments, the first surface is a first side surface and the second surface is a second side surface. In some embodiments, the first surface is an upper surface and the second surface is a side surface. In some embodiments, the first surface is an lower surface and the second surface is a side surface.

[0030] The channels can have a cross-sectional shape that encourages tissue ingrowth. In some embodiments, the cross-sectional shape can be square, triangular, pentagonal, hexagonal, heptagonal, octagonal, rectangular, trapezoidal, oval, torx, star with any number of arms, cloverleaf, leaf with any number of arms, other curved or rectilinear shapes, or combinations thereof. In some embodiments, a group or cluster of channels can have a honeycomb structure.

[0031] In some embodiments, two or more different channel cross sections may be used in a single implant. In some embodiments, some channels are rectangular in cross section and some are hexagonal (creating a honeycomb structure). Any combination that achieves a therapeutic use may be used.

[0032] In some embodiments, the implant may incorporate at least one type of stem cell. In some embodiments, the at least one type of stem cell is a neural stem cell. The neural stem cell is an embryonic stem cell, an iPSC-derived stem cell, a differentiated stem cell, a directly differentiated neural stem cell (e.g., differentiated from skin into neurons without going through a stem cell state), a GFP-expressing neural stem cell, or a combination thereof. In other embodiments, the at least one type of stem cell is a mesenchymal stem cell. The stem cell may be engineered to express BDNF, NT3, GDNF, or a combination thereof.

[0033] Implants can be formed to virtually any length, hi some embodiments, implants can have a length of about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 20 mm, about 30 mm, about 40 mm, about 50 mm, about 2 mm to about 4 mm, about 2 mm to about 10 mm, or about 2 mm to about 20 mm.

[0034] In some embodiments, the implant can be formed with a shape and structure that mimics spinal cord structures. These structures can include, but are not limited to, axonal tracks and channels. Axonal tracks can include the rubrospinal tract, the raphe-anterior raphe-spinal tract, the reticulospinal tract, the propriospinal tract, the spinothalamic tract, and the corticospinal tract.

[0035] In some embodiments, the implant may include entrances and exits for axons within the implant, referred to herein as "channels," which maintain 3D coordinates throughout the lesion site and conform to the natural host structure.

[0036] In some embodiments, the implant can have a modulus of elasticity greater than about 250 kPa, greater than about 200 kPa, greater than about 300 kPa, between about 250 kPa and about 300 kPa, or between about 200 kPa and about 300 kPa.

[0037] In some embodiments, the implant is substantially biostable, meaning that the implant is 80%, 90%, 95%, 99% or completely intact after 3 months, 4 months, 5 months, 6 months, 1 year, or 5 years after implantation.

[0038] In some embodiments, the implant may resist collagen deposition on its surface, ie, the implants described herein may reduce collagen deposition by more than about 50%, more than about 60%, more than about 70%, more than about 80%, more than about 90%, or more than about 95% when compared to implants formed from another polymeric or metallic material.

[0039] In some embodiments, the implants may resist reactive cell deposition (or reduce the size of the reactive cell layer) at the implantation site. In some embodiments, the implants described herein may reduce the size of the reactive cell layer by more than about 20%, more than about 30%, more than about 40%, more than about 50%, or more than about 60% when compared to implants formed from another polymeric or metallic material. In some embodiments, the implants described herein may reduce the size of the reactive cell layer by more than about 20%, more than about 30%, more than about 40%, more than about 50%, or more than about 50%, or more than about 60% when compared to implants formed from agarose.

[0040] In some embodiments, the implant may attract a minimal reactive cell layer after implantation having a thickness of less than about 400 μm, less than about 350 μm, less than about 300 μm, less than about 250 μm, or between about 400 μm and about 200 μm.

[0041] In some embodiments, the implants may reduce glial scar formation at the implantation site by more than about 50%, more than about 60%, more than about 70%, more than about 80%, or more than about 90% when compared to implants formed from another polymeric or metallic material. Glial scar formation (gliosis) is a reactive cellular process, including astrogliosis, that occurs after injury to the central nervous system. In some embodiments, the implants may reduce glial scar formation at the implantation site by more than about 50%, more than about 60%, more than about 70%, more than about 80%, or more than about 50%, or more than about 90% when compared to implants formed from agarose.

[0042] In some embodiments, the implant may reduce glial scar formation at the implantation site by more than about 20%, more than about 30%, more than about 40%, more than about 50%, or more than about 60% when compared to an untreated lesion.

[0043] Compared to agarose implants, the implants of the present invention may result in glial fibers arranged longitudinally in grooves that may potentially support outgrowing axons.

[0044] The implants described herein may be fully vascularized, thereby allowing blood vessels to infiltrate the implant.

[0045] Furthermore, the implants may allow axons to penetrate the implant material. This penetration is in contrast to other implants, such as agarose-based implants, where axons cannot cross the astrocytic scar that develops around those implants. Axons that penetrate the implants described herein may associate with adjacent ensheathing Schwann cells.

[0046] In some embodiments, when the implant incorporates at least one type of stem cell, host axons may enter the stem cell-filled channel and regenerate linearly or in parallel within the channel. In other embodiments, host serotonergic axons may enter the stem cell-filled channel from the rostral aspect of the lesion and regenerate linearly or in parallel within the channel. In some embodiments, serotonergic axons may enter the implant and regenerate linearly or in parallel within the channel even in the absence of stem cells. However, this regeneration may be reduced by about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, or about 40% to about 50% compared to stem cell-containing implants.

[0047] In some embodiments, regenerating host axons can reach the tail of the implant, and in some embodiments, when incorporating at least one type of stem cell, the implant increases axons reaching the tail of the implant by at least about 50%, at least about 80%, at least about 100%, or at least about 120% compared to an implant that does not contain stem cells.

[0048] In some embodiments, the implant may allow motor axons to exit the caudal side of the channel and regenerate into the host spinal cord or peripheral nerve distal to the lesion, hi other embodiments, motor axons may remain detectable up to about 3.5 mm, about 2.5 mm, or about 4.5 mm beyond the lesion.

[0049] In some embodiments, the channels of the implants described herein can accommodate or recruit axons of various diameters. Furthermore, axons that regenerate within the channels can be myelined. In some embodiments, once axons form within the channels, synapses can form between the axons within the channels and the dendrites of transplanted neural stem cells. The synapses can be asymmetric and / or presynaptic boutons containing round vesicles. In some embodiments, at least some of the formed synapses can be excitatory.

[0050] In some embodiments, host axons may regenerate into the channels and form appositional contacts with dendrites of the transplanted neural stem cells.

[0051] In some embodiments, the implants described herein can result in at least partial restoration of motor evoked potential (MEP) responses. Motor evoked potentials are recorded from muscles after direct stimulation of the spinal cord, either magnetically or electrically. In some embodiments, these MEP responses can be in the arms and / or legs, including the fingers and toes. Stem cell-incorporated implants can increase MEP responses by about 4-fold to about 10-fold, about 3-fold to about 10-fold, about 4-fold to about 8-fold, or about 3-fold to about 8-fold when compared to blank implants.

[0052] In some embodiments, the implants described herein can result in at least partial functional improvement of motor behavior after injury. Post-injury motor behavior in rodents is measured using the Basso, Beattie, and Bresnahan (BBB) locomotor scale. The scale (0-21) represents successive stages of recovery and categorizes a combination of joint movement, hindlimb movement, stepping, forelimb and hindlimb coordination, trunk position and stability, paw position, and tail position.

[0053] In some embodiments, after implantation, the implanted channel may be uniformly filled with stem cells. In some embodiments, after implantation, the channel may be uniformly filled with neural stem cells. In some embodiments, the stem cells may occupy the interface between the implant and the recipient. In some embodiments, the stem cell-derived cells in the channel may express neuronal markers, such as, but not limited to, Hu or NeuN. In other embodiments, the stem cell-derived cells in the channel may express oligodendrocyte markers, such as, but not limited to, Olig2, or astrocyte markers, such as, but not limited to, GFAP. In some embodiments, the cells may express two or more of the above.

[0054] Surprisingly, in some embodiments, spinal cord-derived neural stem cell grafts are free of serotonergic neuronal cell bodies.In some embodiments, spinal cord-derived neural stem cell grafts are surprisingly free of serotonergic neuronal cell bodies.

[0055] In some embodiments, recipients implanted with an implant described herein incorporating at least one type of stem cell may exhibit a shorter MEP latency period compared to recipients implanted with an empty implant. In some embodiments, recipients implanted with an implant described herein incorporating at least one type of stem cell may exhibit an MEP latency period that closely resembles that observed in an intact recipient. In some embodiments, the MEP latency period may be between about 9 ms and about 12 ms, between about 8 ms and about 10 ms, between about 8 ms and about 12 ms, between about 9 ms and about 10 ms, or between about 7 ms and about 13 ms.

[0056] As mentioned above, in some embodiments, the implants described herein are printed. Bioprinting functional tissues, typically the face, faces many challenges, including the lack of suitable biofabrication techniques for constructing the complex 3D microarchitectures necessary to guide cell proliferation and promote tissue maturation. Common inkjet or extrusion-based bioprinting approaches use nozzles to deposit materials, enabling the printing of simple 2D structures such as skin and cartilage, and simple 3D structures such as blood vessels, aortic valves, and trachea.

[0057] In some embodiments, the implants described herein can be fabricated using microscale serial projection 3D printing (μCPP). Microscale serial projection printing can fabricate complex 3D architectures using a variety of biomaterials and cells. Such printing can be achieved without scanning in both the X and Y directions (as opposed to nozzle-based approaches). In this way, three-dimensional objects can be fabricated in a single continuous print in the Z direction. In some embodiments, printing the entire implant takes only a few seconds. In some embodiments, the implant can be printed in about 1 second, about 2 seconds, less than about 2 seconds, less than about 3 seconds, less than about 4 seconds, less than about 5 seconds, less than about 10 seconds, less than about 20 seconds, or less than about 30 seconds. In one embodiment, printing an entire 2 mm implant requires only about 1.6 seconds. This printing speed is approximately 1,000 times faster than conventional nozzle printers.

[0058] Using focused light for polymerization produces a printing resolution of 1 μm, a 50-fold improvement over nozzle-based inkjet printing. In inkjet or extrusion-based approaches, artificial interfaces between droplets or lines can compromise mechanical integrity, leading to mechanical failure during or after in vivo application. By providing layer-free resolution in the Z direction, structures do not exhibit these planar artifacts (interfaces) caused by moving a linear stage to a new position. Therefore, the μCPP described herein can improve the mechanical integrity of 3D-printed implants and provide rapid fabrication of complex 3D biomimetic structures with microscale resolution.

[0059] In some embodiments, the implant is printed in a single piece for implantation at the site of spinal cord transection or peripheral nerve injury. In some embodiments, the implant is printed in two or more pieces, thereby allowing damaged but unsevered spinal cords or peripheral nerves to be treated with the implants disclosed herein. In cases where the spinal cord injury (SCI) or peripheral nerve injury is not severed, one or more pieces of the implant can be implanted to surround surviving tissue, and these pieces can be attached to each other using a biologically acceptable adhesive. Thus, any viable tissue can be maintained and regeneration of the recipient's spinal cord or peripheral nerves at the injury site can be promoted.

[0060] Implants are customized for each patient. After imaging the patient's spinal cord or peripheral nerves, a 3D model is created using CAD software. This model is then used to print a patient-specific implant that conforms to and fills the lesion. Therefore, in some cases, it may not be necessary to print the entire implant as designed in Figure 1D. If the lesion is not as large as the entire spinal cord (partial lesion) compared to the model, it will be smaller than what is shown in Figure 1D. The physician makes the final decision on which area, all or the lesioned area, to fill with the implant. For example, the physician may decide to print only the channel portion (excluding the butterfly shape).

[0061] In other embodiments, a 3D printed biomimetic implant is printed based on a spinal cord or peripheral nerve injury, and only a portion of the printed implant is implanted at the injury site. In some embodiments, only the honeycomb portion is implanted.

[0062] The implants described herein demonstrate in vivo stability and can support axonal regeneration and remyelination across sites of severe (complete) SCI. SCI affects over 500,000 people in the United States, resulting in significant psychological and economic costs for both patients and caregivers. Three-dimensional printing using the devices and methods described herein may enable the fabrication of personalized implants that "fit" the precise anatomy of an individual's injury to stimulate, guide, and align axonal regeneration. Furthermore, neural stem cells may be incorporated into the implant to produce implants that further support nerve repair or remyelination.

[0063] In some embodiments, recipient motor axons may regenerate and bridge the intact spinal cord, or peripheral nerve, across the lesion site to the distal spinal cord, or peripheral nerve via the biomimetic implant.

[0064] In other embodiments, the implants and treatment methods of the present invention may support recovery of function in the most difficult model of SCI, complete spinal cord transection.

[0065] In some embodiments, the implants of the present invention, which are a fusion of rapid 3D printing and stem cell biology, may provide treatment of the spinal cord or peripheral nerves by providing patient-specific regenerative therapy.

[0066] In some embodiments, the implant may promote axonal regeneration after spinal cord injury or peripheral nerve injury.

[0067] In some embodiments, the implants may provide for regeneration or remyelination of more than hundreds of injured host axons over distances of 1-20 millimeters or more. In some embodiments, the implants may provide for regeneration or remyelination of more than thousands of injured host axons over distances of 1-20 millimeters or more. In some embodiments, the implants may provide for regeneration or remyelination of host axons over distances of 1-20 mm, 1-10 mm, 1-5 mm, 1-4 mm, 1-3 mm, 1-2 mm, 2-4 mm, 3-4 mm, 2-5 mm, or 3-5 mm. In some embodiments, the implants may support axons that may extend over distances of greater than about 50 mm, greater than about 100 mm, greater than about 150 mm, greater than about 200 mm, or more.

[0068] In some embodiments, implants of the present invention can provide functional improvement to the recipient even after complete spinal cord transection or peripheral nerve transection. In some embodiments, implants of the present invention provide a functional benefit to the recipient after stem cell transplantation, resulting in "splicing" the damaged circuit, with recipient axons penetrating and synapsing with neurons in the graft, and axons from the graft then extending from the lesion site and synapsing with neurons in the recipient's spinal cord or peripheral nerve cells caudal to the injury.

[0069] In some embodiments, the implants of the present invention can optimize the functional availability of axons emerging from the neural stem cell implant at the lesion site, hi some embodiments, the implants of the present invention can align connected circuits with their correct caudal white matter processes.

[0070] In some embodiments, the implants of the present invention may provide a "custom fit" implant for an individual patient's lesion.

[0071] Also described are methods of manufacturing the implants described herein. The methods can include scanning an area requiring treatment and printing an implant described herein. The printing can be by 3D printing.

[0072] Additionally, methods for treating conditions using the implants described herein are described. Conditions may include, but are not limited to, neuropathy, spinal cord injury, complete motor spinal cord injury, partial motor spinal cord injury, peripheral nerve injury, bowel dysfunction, incontinence, impotence, other sexual dysfunction, pain, numbness, neuropathy, poor temperature regulation, and the like, or combinations thereof. Some of these conditions are sequelae of spinal cord injury, and therefore, treating the injury site and restoring function at the injury site will treat one or more of the sequelae.

[0073] In one embodiment, a method of treating nerve damage is described that may include scanning an area requiring treatment, printing an implant as described herein, implanting the implant into a location within the area, and treating the nerve damage.

[0074] In one embodiment, a method of treating spinal cord injury is described that may include scanning an area requiring treatment, printing an implant as described herein, implanting the implant into a location within the area, and treating the spinal cord injury.

[0075] In some embodiments, methods of treating complete motor spinal cord injury are described. The method for treating complete motor spinal cord injury may include scanning an area of the spinal cord requiring treatment, printing an implant as described herein, implanting the implant into a location within the area, and treating the motor spinal cord injury.

[0076] In some embodiments, methods are described for treating paralysis that may include scanning an area of the spinal cord requiring treatment, printing an implant as described herein, implanting the implant into a location within the area, and treating the paralysis.

[0077] In some embodiments, a method for treating bowel dysfunction resulting from spinal cord injury is described. The method for treating bowel dysfunction resulting from spinal cord injury can include scanning an area of the spinal cord requiring treatment, printing an implant as described herein, implanting the implant into a location within the area, and treating bowel dysfunction resulting from spinal cord injury.

[0078] In some embodiments, a method for treating impotence resulting from spinal cord injury is described. The method for treating impotence resulting from spinal cord injury can include scanning the area of the spinal cord requiring treatment, printing an implant as described herein, implanting the implant into a location within the area, and treating impotence resulting from spinal cord injury.

[0079] In some embodiments, methods for treating pain are described that may include scanning an area of the spinal cord requiring treatment, printing an implant as described herein, implanting the implant into a location within the area, and treating the pain.

[0080] In some embodiments, methods for treating peripheral nerve injury are described. Methods for treating complete or partial peripheral nerve injury may include scanning the area of the peripheral nerve site requiring treatment, printing an implant as described herein, implanting the implant into position within the area, and treating the peripheral nerve injury.

[0081] In some embodiments, the location can be a spinal cord lesion.

[0082] In some embodiments, the location can be a peripheral nerve lesion.

[0083] In some embodiments, the treatment method may further include exposing the individual to other treatment modalities. These therapies may include training devices or systems configured to physically train the subject, thereby providing additional neural signals to parts of the subject's body that have been impaired by the injury. The training devices may use robotics, exoskeletons, treadmills, canes, walkers, crutches, body weight support systems, physical therapy, or combinations thereof, to assist in training.

[0084] In some embodiments, the method of treatment may further include growing axons through the implant channels.

[0085] Kits are also described, which may include the implant and instructions for use in a combined container.

[0086] Some kits may include a scan of the area requiring treatment in an integrated container and instructions for use.

[0087] Other kits may include a scan of the area requiring treatment, the polymer needed to print the implant, and instructions for use in an integrated container.

[0088] Other kits may include a scan of the area requiring treatment, PEGDA and GelMa for printing the implant, and instructions for use in a single container.

[0089] Example 1 The spinal implant was designed using a rat spinal cord as a template.

[0090] The implant material used in this example: PEGDA (Mn = 700 Da) was purchased from Sigma-Aldrich (USA). Gelatin methacrylate (GelMa) was synthesized as previously reported (Soman, P. et al., Biotechnol Bioeng 110:3038-3047, 2013). The photoinitiator phenyl-2,4,6-trimethylbenzoylphosphinate lithium (LAP) was synthesized as previously reported (Fairbanks, BD et al., Biomaterials 30:6702-6707, 2009). The matrix material used to print the implants was made by mixing 7.5% (w / v) GelMa, 25% (v / v) PEGDA, and 0.225% (w / v) LAP in Dulbecco's phosphate-buffered saline (DPBS).

[0091] Implant 3D Printing Used in This Example: The 3D bioprinter (μCPP) described herein contains the following six components, as shown in Figure 1A: (1) a UV LED light source (365 nm) for photopolymerization; (2) a digital micromirror array device (DMD) chip (Texas Instruments) consisting of 1920 × 1080 micromirrors for optical pattern generation; (3) projection optics for imaging the optical pattern on the DMD chip onto a fabrication plane on a stage; (4) an automated stage to hold the monomer solution for fabrication; (5) a digital camera for real-time monitoring and imaging of the fabrication process; and (6) a computer to coordinate the UV light source, DMD chip, stage, and camera for the 3D printing process.

[0092] The rat spinal cord implant printing used in this example: Digital images of the core (representing the spinal cord's gray matter) and shell (representing the spinal cord's white matter) were generated by processing cross-sectional images of the spinal cord, and these were later incorporated into the DMD chip to control the micromirrors during the printing process. Channels (200 μm in diameter) were incorporated into the shell to provide linear or parallel guidance for axonal regeneration. The core was designed as a solid block of GelMa, 25% (v / v) PEGDA, and 0.225% (w / v) LAP to enhance the mechanical strength of the printed implant. The matrix material monomer solution was placed in a container with a 2 mm PDMS spacer to control the z-axis height of the printed implant. The sequential printing process was initiated using in-house developed software to control the 3D printer. The implant was printed in two stages, each 0.8 seconds long: one for the shell image and one for the core image. The printed implant was then removed from the reservoir and rinsed three times with sterile DPBS and antibiotics (1% Pen-Strep).

[0093] Human spinal cord implant printing used in this example: A typical chronic spinal cord injury was modeled using cervical MRI scans. The lesion was tracked and a 3D spinal cord computer-aided design (CAD) model was used to match the dimensions of the human injury. The 3D model was then sliced along the length of the spinal cord into a series of digital masks, which were then sequentially integrated into the DMD chip. By dynamically modifying the digital masks as the stage moved, patient-specific spinal cord implants were created using the above method.

[0094] Fabrication of templated agarose scaffolds used in this example: Multicomponent fiber bundle (MCFB) templates were fabricated from 200 μm diameter polystyrene fibers (Paradigm Optics, Vancouver, WA) arranged in a hexagonal close-packed array separated by a continuous matrix of poly(methyl methacrylate) (PMMA). They were arranged in a honeycomb array with 66 μm spacing to create a final implant with a 66 μm wall size and 200 μm channel diameter. The bundles were extruded and fused together so that the polystyrene fibers were oriented parallel to the longitudinal axis of the bundle. The multicomponent fiber bundle template was trimmed to a length of 2 mm and a cross-sectional width and depth of 1.5 mm. 1.5 mm long polystyrene end caps were attached to the fiber bundle ends using cyclohexane to anchor the polystyrene fibers, forming an external rigid multicomponent fiber bundle template. Six such multicomponent fiber bundle units were then aligned in series with two polystyrene side caps, aggregating into a linear template array. The poly(methyl methacrylate) matrix was then selectively removed by immersion in 99.7% propylene carbonate (Sigma-Aldrich) three times, followed by rinsing with 95% ethanol and distilled water. Ultrapure agarose (30 mg / ml, Sigma-Aldrich) was dissolved in distilled water at 100°C and then cooled to 65°C. The multicomponent fiber bundle template was immersed in the agarose solution and centrifuged (300 rpm for 30 seconds) to permeate the agarose through the packed polystyrene fiber array. The agarose cast was then allowed to gel at room temperature, trimmed, and immersed in 99% tetrahydrofuran (Sigma-Aldrich) for 24 hours at room temperature. This process was repeated twice to remove the polystyrene mold, yielding individual free-floating agarose scaffolds. The scaffolds were collected and washed sequentially with acetone, 95% ethanol, and three cycles of sterile water. They were stored in sterile water at room temperature until use.

[0095] Preparation of E14 neural stem cells used in this example: Briefly, spinal cords from GFP-expressing E14 F344 embryos were dissected and the meninges were removed. The tissue was trypsinized for 15 minutes and then centrifuged at 2500 rpm at room temperature. The tissue was resuspended in NeuroBasal medium (Gibco) containing 2% B27 (Gibco), and the spinal cord tissue was gently triturated using successively smaller fire-polished Pasteur pipettes. The cells were then centrifuged at 2500 rpm for 2 minutes, resuspended in NeuroBasal medium containing B27, and filtered using a 40 μm cell filter strainer.

[0096] The surgical procedures used in this example strictly adhered to NIH guidelines for the care and safety of laboratory animals. Implants were implanted in a complete transverse section at the T3 spinal cord level. Briefly, animals were deeply anesthetized, and a T3 laminectomy was performed. Subsequently, the spinal cord was transected using a combination of microscissors and microsuction. A 1.8 mm block was removed, and a 2 mm-long implant was implanted, thus firmly holding the implant between the transected segments of the spinal cord. Group 1 (n = 14) received an empty agarose scaffold, Group 2 (n = 14) received an empty 3D-printed implant, and Group 3 (n = 14) received a 3D-printed implant incorporating E14 neural stem cells suspended in a fibrin matrix containing a four-component growth factor cocktail. The implants were treated with 50 ng / μL BDNF (Peprotech) to support neural stem cell survival, 10 ng / μL VEGF (Peprotech) and 10 ng / μL bFGF (Peprotech) to promote angiogenesis, and 50 μM MDL28170 (Sigma), a calpain inhibitor for neuroprotection. Group 4 (n=8) underwent a sham surgery in which the injury was treated but no implant was implanted. Group 5 (n=8) received implants of multipotent neural progenitor cells derived from rat E14 spinal cord as previously described (Lu, P. et al. Cell 150:1264-1273, 2012). The cells were suspended in the same fibrinogen / thrombin matrix with the growth factor cocktail described above. After implantation, the back muscles and skin were sutured, and antibiotics and analgesics were administered.

[0097] The microchannels (200 µm in diameter) were designed to guide and align axons from their transection point above the injury to their correct reentry point below the injury where they re-enter the intact spinal cord (Figure 1A-E). The inner core region typically does not contain axons protruding below the injury site. Therefore, this component of the implant was designed as a solid region to enhance the implant's structural support. Previous studies using agarose microchannel implants have shown that 80% of axons entering the lesion site are guided by the implant and bridged to the other side of the lesion. However, agarose induces a foreign body response consisting of a collagen-based reactive cell layer that weakens and traps axons within the implant, preventing them from exiting the channel. Therefore, the implant was fabricated from a degradable material that can reduce the reactive cell layer to reduce the foreign body response, thereby allowing host axons to better penetrate and traverse the lesion. A combination of two biocompatible materials, polyethylene glycol diacrylate (PEGDA) and gelatin methacrylate (GelMa), was used as the implant material. PEGDA is non-adhesive to cells, and therefore, GelMa, a photopolymerizable modified collagen that retains cell-binding ligands and matrix metalloproteinase (MMP) degradation sites, was added to potentially enhance long-term cell viability. Because a mismatch in mechanical properties between the implant and the host can lead to compression or tearing at the spinal cord interface, causing integration failure, the concentration of each material and the crosslinking density of the printed implant can be designed to mimic the mechanical properties of native spinal cord tissue.

[0098] Dynamic mechanical analysis (DMA) was used to measure the elastic modulus of the 3D-printed PEGDA / GelMa implants. The elastic modulus of the bioprinted implants used for implantation was in the range of 260 kPa–300 kPa, in accordance with the elastic modulus of the native spinal cord, 200–600 kPa (Figure 2).

[0099] Immunolabeling was performed. Spinal cords were sectioned with a cryostat set at 20 μm intervals and processed for: 1) GFP labeling to assess the survival and differentiation of transplanted cells and axonal outgrowth (GFP rabbit polyclonal, Invitrogen, 1:500 dilution); 2) neuronal markers including Hu for young neurons (human polyclonal, 1:500 dilution), NeuN for mature neuronal nuclei (mouse monoclonal, Abcam, 1:500 dilution), and MAP-2 for mature neurons (mouse monoclonal, BD Biosciences, 1:500 dilution), Neurofilament 200 (mouse monoclonal, Millipore, 1:500 dilution) to label axons, serotonin (5HT, goat polyclonal, ImmunoStar, 1:500 dilution) for mature neurons and axons, glial fibrillary acidic protein (GFAP, chicken polyclonal, Millipore, 1:500 dilution) for astrocytes, and Olig2 (Olig2, mouse monoclonal, IBL, 1:200 dilution) for oligodendrocytes. 3) S100 (rabbit polyclonal, Dako, 1:500 dilution) to label Schwann cells. 4) Collagen type IV (rabbit polyclonal, Biogenex, 1:500 dilution). Sections were incubated overnight in primary antibodies at room temperature, followed by 3 hours in Alexa 488-, 594-, or 647-conjugated goat or donkey secondary antibodies (1:250, Invitrogen) at room temperature. The thickness of the reactive cell layer was measured in Nissl-stained sections at 200x total magnification (eight sections were quantified per animal, and results were expressed as mean ± SEM). GFAP immunoreactivity was quantified as the mean gray value per pixel measured at the recipient spinal cord-implant interface on GFAP-immunolabelled sections (eight sections were quantified per animal, and results were expressed as mean ± SEM). 5HT motor axon quantification was performed using 200x magnification images of 400 μm sections of the tail of the channel (axons were manually counted and eight sections were quantified per animal). The above antibodies were used to quantify stem cell differentiation within the channel. Each slide was counterlabeled with DAPI and GFP, and cells were manually counted. Each cell type number was normalized to the total DAPI / GFP-labeled nuclei in the channel (8 sections were quantified per animal).Quantification was performed using ImageJ.

[0100] Statistical analysis: Comparisons between two groups were tested by two-tailed Student's t-test (JMP software) with a specified significance level of P < 0.05. Multiple group comparisons were tested by one-tailed ANOVA (JMP software) with a specified significance level of P < 0.05, followed by post-hoc analysis using Tukey's test.

[0101] Electron Microscopy - Detailed analysis of synaptogenesis and axonal myelination was performed using electron microscopy as follows: subjects were perfused with 4% paraformaldehyde and 0.25% glutaraldehyde, and spinal cords were postfixed in 1% osmium tetroxide, dehydrated, and explanted in durcupan resin. For general morphology, 0.5 μm semithin sections were stained with toluidine blue. 60 nm sections were then cut using an ultramicrotome and visualized using an FEI 200KV Sphera microscope at the UCSD CryoElectron Microscopy Core Facility.

[0102] Scanning Electron Microscopy Imaging - Patient-specific spinal implants were imaged using a scanning electron microscope (SEM, Zeiss Sigma 500). The implants were dehydrated in a series of ethanol baths and dried in a supercritical point dryer (Tousimis AutoSamdri 815A) before being sputter-coated with iridium for 7 seconds at a deposition current of 85 mA using an Emitech K575X. After sputter coating, the implants were imaged at 5 kV using a Zeiss Sigma 500 SEM.

[0103] Functional Analysis—BBB open field 21-point locomotor rating scale was assessed weekly by two independent observers blinded to the identity of the groups.

[0104] Electrophysiology - MEPs were measured in the hindlimbs. Briefly, animals were anesthetized with propofol (100 mg / kg, PropoFlo Abbott). Transcranial electrophysiology (9 mV, 1 ms pulse width using a DS3 constant current insulated stimulator (Digitimer, Welwyn Garden City, UK)) was performed using two 30 G stainless steel stimulating electrodes placed percutaneously. MEPs were recorded using ring electrodes placed in both hindlimbs until three to five highest (stable) recorded potentials were similar. MEPs were recorded 26 weeks after implantation.

[0105] In some embodiments, an advantage of 3D bioprinting is the ability to rapidly print implants of different sizes (Figure 17A-B) and unusual shapes to fit individual patients' lesion sites, as confirmed by preoperative MRI scans. As shown in Figures 3A-E, implants were printed to fit the precise shape and size of the patient's chronic lesion cavity.

[0106] Bioprinted implants were implanted into rat spinal cords at a complete T3 transection. This is the most severe model of SCI and the most challenging model for studying spinal cord regeneration. It is also a model for studying axon regeneration because, unlike contusion injury, which has a rim of spare tissue with surviving axons, axons are severed at the time of injury, making it difficult to determine whether the axons observed are truly regenerating axons or stagnant or sprouting axons. Nineteen Fischer 344 rats underwent a complete T3 spinal cord transection, and 2-mm-long implants were immediately placed at the lesion site. Eight control animals had lesions only. After 4 weeks, the spinal cords were removed and implant structure, biocompatibility, and axon regeneration / remyelination were assessed. Findings were compared with animals previously receiving molded agarose implants with the same lesion and survival time.

[0107] Four weeks after implantation, the 3D-printed PEGDA / GelMa implants maintained their structural integrity. The implant channels and solid core maintained their pre-implant structure without fracture or deformation in all animals. Implant biodegradation was not yet evident at this 4-week time point. Earlier efforts using other implant materials, such as hyaluronic acid, resulted in more rapid implant degradation and structural collapse (Figure 18A). Implant degradation was characterized over 6 months, with a 44 μm reduction in wall thickness, representing 66% preservation of the structure (Figure 19). Maintenance of implant structure is believed to be essential for maintaining physical support across the lesion site and supporting, organizing, and aligning regenerating axonal growth.

[0108] Anatomical analysis after 6 months showed that all implants retained their 3D architecture (Figure 15A). However, implant wall thickness decreased by 49% compared to their pre-implantation size, suggesting slow degradation over time. Among animals implanted with empty implants, relatively few recipient neurofilament-labeled axons regenerated into the implant (118 ± 8), similar to the number observed 4 weeks after implantation (97 ± 8 axons). In animals implanted with empty implants, recipient axons did not regenerate beyond the implant into the distal recipient spinal cord. Among animals implanted with 3D biomimetic PEGDA / GelMa implants incorporating neural stem cells, the transplanted cells survived for 6 months and completely filled all channels (Figure 15A). Nestin labeling was not detected, indicating the completion of maturation of the transplanted neuronal stem cells, and Ki67 labeling was also not detected, indicating the completion of cell division through the graft. As observed with the 4-week implant, 5HT-immunoreactive axons entered the implant. 87 ± 5 serotonergic axons reached the caudal end of the neural stem cell-incorporated channel and continued to regenerate into the caudal spinal cord, similar to the number of axons observed 4 weeks after implantation (Figure 14B). This finding suggests that serotonergic axon regeneration into the implant is complete by 4 weeks. Recipient corticospinal motor axons anterogradely labeled by injection of an AAV2 vector expressing red fluorescent protein (RFP) into the motor cortex also regenerated into the stem cell-incorporated implant (Figure 15B), extending up to the implant midpoint, a distance of 1 mm. Corticospinal axons formed putative bouton-like structures on NeuN-labeled neurons within the implant channel (Figure 15C). Furthermore, graft-derived GFP-labeled axons projected from the implant and entered the recipient spinal cord caudal to the lesion, forming putative bouton-like structures on recipient neurons in the spinal cord caudal to the lesion (Figure 15D-E). The amount of graft-derived axonal outgrowth from the implant into the distal recipient spinal cord (FIG. 15D) greatly exceeded the number of recipient serotonergic axons regenerating beyond the implant.This finding suggests that, if present, the restored neural relay across the lesion site may be mediated by host axons synapsing on transplanted neural stem cells and by stem cell-derived axons extending to the distal host spinal cord.

[0109] Attenuation of the reactive cell layer was present in animals receiving 3D-printed implants compared to the template agarose scaffolds, characterized by reduced collagen deposition (Figure 4B) and reduced granulation tissue (Figure 5A). The reactive cell layer was 340 ± 52 µm thick, a significant 35% reduction compared to the agarose scaffolds (P < 0.05; Figure 5A). The astrocytic response was also attenuated by the 3D-printed implants: in control lesion subjects, astrocytes became reactive and were "detached" from the lesion site (Figure 6A). In animals with agarose scaffolds, the astrocytic wall was still present and peeled off the scaffold from the recipient spinal cord (Figure 6B). In contrast, the 3D-printed implants showed attenuation of the astrocytic scar thickness and scar reorganization, with uninterrupted continuity from the recipient spinal cord to the implant channel (Figure 6C). In some embodiments, astrocyte processes transitioned from forming walls perpendicular to the host interface to forming strands that penetrated linearly through the implant, resulting in association with regenerating host axons. 3D-printed implants demonstrated a 66% reduction in astrocyte immunoreactivity compared to agarose scaffolds and a 97% reduction compared to lesion-only animals (P<0.05; Figure 6D). The implants were readily and extensively vascularized along their entire length (Figures 7A-B).

[0110] As the thickness of the reactive cell layer decreased, host axons approaching the 3D-printed implant aligned along the rostral-caudal (descending) axis of the spinal cord and easily penetrated the implant's channels without deflection. This contrasted with the frequent misalignment and deflection of axons at the interface between the agarose scaffold and the host (Figure 8A-B). Host Schwann cells from the peripheral nervous system migrated into the implant and ensheathed or remyelinate the regenerating host axons (Figure 9A-C).

[0111] In some embodiments, the spinal cord repair implants described herein can incorporate cells capable of enhancing regeneration or remyelination. Thus, 3D printed implants were loaded with GFP-expressing rat neural stem cells harvested from the spinal cord of embryonic day 14 Fischer 344 rats. A total of 3 x 10 6 Cells were loaded into the implant in a volume of 8 μl via direct injection. A total of 14 rats underwent complete T3 spinal cord resection, a 1.8 mm-long spinal cord segment was removed, and a 2 mm-long 3D-printed implant loaded with neural stem cells was implanted. The animals survived for 4 weeks and were then sacrificed to assess implant integrity, cell survival, and host axon regeneration and remyelination.

[0112] In some embodiments, stem cells survived and filled the implant channels in all transplanted animals (Figure 10A, Figures 20A-D). Neural stem cells were also present at the interface between the implant and the recipient spinal cord without distorting the structure of the implant or the recipient spinal cord (Figures 20A-D). Of the samples tested, 47 ± 2% of the transplanted stem cells expressed the early neuronal marker Hu (Figure 21A), 20 ± 3% of the transplanted cells expressed the mature neuronal marker NeuN (Figure 21B), 11 ± 2% of the cells expressed the oligodendrocyte marker Olig2 (Figure 21C), and 21 ± 3% of the cells expressed the astrocyte marker GFAP (Figures 21D and 22). The stem-state marker nestin was not detected.

[0113] Recipient axons readily penetrated the implant (distinguished from transplant-derived axons by the lack of GFP reporter expression) (Figure 10B). Many recipient long-distance serotonergic axons also readily penetrated stem cell-loaded 3D-printed implants and linearized along the channel direction (Figure 10C). Stem cell grafts failed to linearize the penetrating axons (Figure 23). 5HT axons were induced to regenerate to the caudal end of the implant (Figure 10E). In contrast, most serotonergic axons reached the tail of empty implants (lacking stem cell loading) or stem cell grafts (Figure 24A-B). We quantified an average of 85 ± 21 serotonergic axons within the caudal 400 µm of stem cell-loaded channels per implant per animal, compared with 11 ± 5 axons in empty 3D-printed implants. In animals with stem cell grafts lacking implants, an average of 8 ± 4 axons reached the distal end of the lesion site, a 10-fold reduction in axon numbers compared to implants containing stem cell grafts (P < 0.05 ANOVA, P < 0.05 post-hoc Tukey's test comparing implants with vs. without stem cells; Figure 10F). Thus, 3D-printed implants containing neural stem cells may, in some embodiments, enhance host axon regeneration to the caudal end of the lesion site. In other embodiments, this enhancement is significant and substantial.

[0114] In some embodiments, recipient serotonergic motor axons regenerated completely through the lesion site / implant and re-entered the caudal spinal cord (Figure 10G). Because neural stem cells in the implant were not immunolabeled for 5HT (serotonin) and these axons were not labeled for GFP (Figure 25), serotonergic axons located in the recipient spinal cord caudal to the lesion were not derived from the graft. At a distance of 2 mm caudal to the implant, serotonergic axons were detected in the white and gray matter of the recipient spinal cord, frequently branching into the gray matter (Figure 26A-C). Long tracts of recipient serotonergic axons were detected up to 3.5 mm beyond the lesion site (Figure 10H), but not beyond, further supporting the fact that axons were regenerating. In some embodiments, the present implants and devices can provide recipient motor axon regeneration within and beyond implants implanted at complete spinal cord transection sites. Regeneration was further evidenced by the presence of GAP43 immunolabeled axons in the channels and recipient caudal spinal cord (FIGS. 27A-B).

[0115] In some embodiments, a barrier to tissue engineering is organ vascularization. Toluidine blue and electron microscopy analysis demonstrated extensive vascularization within the implant channels described herein (Figures 28A-B). Platelet-derived growth factor receptor (PDGFR) immunolabeling around these blood vessels confirmed the presence of pericytes and restoration of the blood-brain barrier (Figure 29). Electron microscopy analysis of axons within channels filled with neural stem cells demonstrated a range of axon caliber and myelination status, from small, unmyelinated axons (<1 μm diameter) to heavily myelinated axons (1-3 μm, Figure 11). Toluidine blue staining indicated that oligodendrocytes had myelined those axons (Figure 12). Because the implants incorporated neural stem cells expressing the mature neuronal marker NeuN, the potential for synapse formation existed between regenerating host axons and neurons in the implant channels. In some embodiments, asymmetric synapses were readily observed receiving input from axons containing round synaptic vesicles typical of excitatory synapses (Figure 13). Recipient serotonergic axons regenerating into channels could be closely associated with dendrites of stem cell-derived neurons, as identified by colabeling of MAP2 and GFP (Figure 14), also suggesting synaptogenesis.

[0116] Functional and behavioral outcomes were measured using two independent studies. At 26 weeks after implantation, electrophysiological studies were performed by applying transcranial electrical stimulation to the motor cortex and recording motor-evoked potentials (MEPs) from the hind limbs. MEPs can be used to test electrophysiological functional recovery (in both humans and animals) by testing supraspinal control of the brain in the peripheral nervous system and by recording EMG signals from muscles. Rats implanted with 3D-printed implants incorporating neural stem cells 26 weeks after injury showed recovery of MEP responses that were lost upon resection of the spinal cord at the C8 spinal cord level (above the implantation site; T3). These data indicate that muscle activity in the hind limbs was generated by synaptic transmission from the host across the implant (Figures 15A-E, 31). Thus, in some embodiments, the described implants can provide synaptic transmission from the host across the implant to provide muscle activity in the hind limbs.

[0117] Because the hind limbs were denervated and the animals were not weight-bearing, muscles atrophied, leaving fewer muscle units available to respond. This explains the size difference between intact and experimental animals (Figures 15A-E). Consistent with this observation, MEP amplitudes were significantly greater in animals with empty implants than in animals with empty implants (p<0.05, Figure 15E). Furthermore, the latencies (time to maximum amplitude) of recorded MEPs were shorter in stem cell implants and closer to the observed latencies in intact animals (ANOVA p<0.01, Figure 3C).

[0118] To determine the extent of motor recovery with the 3D biomimetic PEGDA / GelMa implants, animals were assessed using the Beattie Basso Bresnahan (BBB) locomotor activity scale for 6 months until behavior plateaued and stabilized. Animals receiving neural stem cell-loaded implants showed significant functional recovery compared to animals with empty implants.

[0119] During the first 4 weeks after injury, hindlimb locomotion was impaired (e.g., severely) in both lesioned controls and transplanted patients. At 5 weeks, recipients of NSC-incorporated implants began to show improvement on the BBB scale, reaching a level of 7, showing movement for each joint of the hindlimb compared to minimal, if any, movement in lesioned controls (repeated measures ANOVA p<0.01; individual time points *p<0.01; Figure 16).

[0120] Functional scores reached a mean value of 6.6 ± 0.5 points (+SEM) on the BBB scale in animals that received neural stem cells in implants 6 months prior, in contrast to a mean score of 0.3 ± 0.2 points in empty implant controls, indicating movement around each joint of the hindlimb, reflecting inconsistent movement around only one joint (*p<0.01, repeated measures ANOVA; individual time points and post-hoc Tukey's t-test; Figure 15F). The formation of neural relays was further examined by electrophysiological transmission across the complete transverse section by measuring myogenic MEPs from the hindlimb in response to electrical stimulation of the brain (Figure 15G, Figure 16A-C).

[0121] Six months after injury, rats implanted with 3D biomimetic PEGDA / GelMa implants incorporating neural stem cells showed recovery of motor-evoked responses, whereas animals implanted with empty implants exhibited responses within the baseline noise range (p<0.01, t-test; Figures 15G and 16D). Resection of the spinal cord at the C8 level (above the implant site) resulted in the loss of all evoked potentials in the hind limbs (Figures 16A-C), confirming the formation of a new electrophysiological relay across the lesion.

[0122] This study demonstrates the use of rapid 3D printing to print biomimetic central nervous system structures. These implants can be easily customized to fit the shape and length of specific lesions. 3D-printed PEGDA / GelMa implants can maintain their structure for at least 26 weeks in vivo. Furthermore, the described printed implants can support the engraftment of neural stem cells. Furthermore, the described printed implants can support the formation of new synapses. In some embodiments, the implants are fully vascularized, providing sufficient availability of blood, oxygen, and nutrients to support consistent cell and axonal survival.

[0123] The foregoing disclosure is of exemplary embodiments. Those skilled in the art will appreciate that the devices, techniques, and methods disclosed herein describe representative embodiments that will function well in the practice of the present disclosure. However, those skilled in the art, in light of this disclosure, will recognize that many changes can be made to the specific embodiments disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0124] Unless otherwise indicated, all numbers expressing properties, such as quantities of ingredients, molecular weights, reaction conditions, and the like, used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the following specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the scope of the claims and the application of the doctrine of equivalents, each numerical parameter should be construed in light of at least the number of reported significant digits and ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. However, such numerical values inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0125] Unless otherwise indicated herein or clearly contradicted by context, the terms "a," "an," and "the" and similar referents as used in the context of describing the present invention (particularly in the context of the appended claims) should be construed to cover both the singular and the plural. The recitation of ranges herein is merely intended to serve as a shorthand method of individually referring to each separate value within the range. Unless otherwise indicated herein, the separate values are incorporated herein as if they were individually listed herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "etc.") provided herein is merely to better clarify the invention and does not otherwise limit the scope of the invention as claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0126] Although the present disclosure supports definitions that refer only to alternatives and "and / or," use of the term "or" in the claims will be used to mean "and / or" unless expressly indicated to refer only to alternatives or unless the alternatives are mutually exclusive.

[0127] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referenced and claimed individually or in any combination with other members within the group or with other elements described herein. It is anticipated that one or more group members may be included in, or deleted from, a group for reasons of convenience and / or patentability. When such inclusion or deletion occurs, the specification is deemed to contain the group as modified herein, thereby satisfying the written description of all Markush groups used in the appended claims.

[0128] Preferred embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations of these preferred embodiments will be apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect those skilled in the art to employ such variations as they see fit, and the inventors intend to practice the invention otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, this invention includes any combination of the above-described elements in all possible variations thereof unless otherwise indicated herein or clearly contradicted by context.

[0129] Certain embodiments disclosed herein may be further limited in the claims using the language "consisting of" or "consisting essentially of." When used in a claim, whether added at the time of filing or by amendment, the transitional term "consisting of" excludes any element, step, or ingredient not recited in the claim. The transitional term "consisting essentially of" limits the claim to specific materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the invention so claimed are essentially or explicitly described herein and enabled herein.

[0130] Furthermore, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, and not of limitation, alternative configurations of the invention may be utilized in accordance with the teachings herein. Accordingly, the invention is not limited to that precisely as shown and described.

[0131] (Addendum) (Appendix 1) a three-dimensional (3D) implant comprising a first end and a second end, the implant comprising a core and a shell, the three-dimensional (3D) implant mimicking the structure of the injury site; at least one channel in the shell beginning at the first end and terminating at the second end; at least one type of stem cell contained in said at least one channel; Biomimetic implants for spinal cord or peripheral nerve injuries, including:

[0132] (Appendix 2) 2. The implant of claim 1, wherein the implant is manufactured by 3D printing.

[0133] (Appendix 3) 2. The implant of claim 1, wherein the at least one type of stem cell is a neural stem cell.

[0134] (Appendix 4) 4. The implant of claim 3, wherein the neural stem cells are embryonic stem cells, iPSC-derived stem cells, directly differentiated neural stem cells, or a combination thereof.

[0135] (Appendix 5) 2. The implant of claim 1, wherein the at least one type of stem cell is a mesenchymal stem cell.

[0136] (Appendix 6) 2. The implant of claim 1, wherein the stem cells are engineered to express BDNF, NT3, GDNF, or a combination thereof.

[0137] (Appendix 7) 2. The implant of claim 1, wherein the three-dimensional printed implant comprises polyethylene glycol diacrylate or gelatin methacrylol, or a combination thereof.

[0138] (Appendix 8) 2. The implant of claim 1, wherein the implant is a biomimetic for the spinal cord.

[0139] (Appendix 9) 2. The implant of claim 1, wherein the implant is a biomimetic for a peripheral nerve.

[0140] (Appendix 10) 10. The implant of claim 1, wherein the channel is linear.

[0141] (Appendix 11) 10. The implant of claim 1, wherein the channels are parallel to one another.

[0142] (Appendix 12) 2. The implant of claim 1, wherein the channel guides regenerating axons from the first end to the second end.

[0143] (Appendix 13) 2. The implant of claim 1, wherein the implant comprises two or more channels having hexagonal cross-sections clustered in a honeycomb structure.

[0144] (Appendix 14) 1. A method of treating nerve damage in a host in need thereof, comprising: Implanting the biomimetic implant described in Appendix 1 at the location requiring treatment; allowing cellular regeneration at the site of injury; A method comprising:

[0145] (Appendix 15) 15. The method of claim 14, wherein the nerve injury is a spinal cord injury, a motor-complete spinal cord injury, a motor-incomplete spinal cord injury, or a peripheral nerve injury.

[0146] (Appendix 16) 16. The method of claim 15, wherein the nerve injury is a spinal cord injury.

[0147] (Appendix 17) 16. The method of claim 15, wherein the nerve injury is a peripheral nerve injury.

[0148] (Appendix 18) 15. The method of claim 14, further comprising providing physical therapy to the host.

[0149] (Appendix 19) scanning the location of the recipient's spinal cord or peripheral nerve requiring treatment to determine the area of said injury; three-dimensionally printing the implant to surround the area of the injury; 2. A method for producing a biomimetic implant according to claim 1, comprising:

Claims

1. a layerless three-dimensional (3D) printed implant body including a first end and a second end, the implant body being formed into a biomimetic shape to mimic the structure of a nerve injury site or spinal cord injury site with respect to axonal tissue of a host; a plurality of linear channels within the implant body beginning at the first end and terminating at the second end, the plurality of linear channels mimicking axonal tissue of the host; Including, The implant body herein comprises polyethylene glycol diacrylate (PEGDA) or gelatin methacrylate (GelMa), the plurality of linear channels comprises a channel wall thickness of up to 100 micrometers, and the plurality of linear channels retain their pre-implantation structure for at least four weeks after implantation. Biomimetic neural or spinal implants.

2. The implant of claim 1 , wherein the plurality of linear channels are oriented parallel to host axons and extend continuously from the first end to the second end.

3. The implant of claim 1 , wherein the plurality of linear channels are microchannels.

4. The implant of claim 1 , wherein the plurality of linear channels comprises a diameter of about 200 micrometers.

5. 10. The implant of claim 1, wherein the plurality of linear channels are spaced approximately 66 micrometers apart.

6. The implant of claim 1 , wherein the plurality of linear channels comprises hexagonal cross sections clustered in a honeycomb structure.

7. The implant of claim 1 , wherein the implant connects severed segments of the spinal cord injury site.

8. The implant of claim 1 , wherein the plurality of linear channels connect severed segments of the spinal cord injury site.

9. The implant of claim 1 , further comprising at least one type of stem cell contained in said plurality of linear channels, said at least one type of stem cell being a neural stem cell.

10. 10. The implant of claim 9, wherein the stem cells are engineered to express BDNF, NT3, GDNF, or a combination thereof.

11. 10. The implant of claim 9, wherein the neural stem cells are embryonic stem cells, iPSC-derived stem cells, directly differentiated neural stem cells, or a combination thereof.

12. The implant of claim 1 , wherein the implant comprises BDNF, NT3, GDNF, or a combination thereof.

13. 10. The implant of claim 1, wherein the implant further comprises lithium phenyl-2,4,6-trimethylbenzoylphosphinate.

14. The implant of claim 1 , wherein the implant comprises about 25% (v / v) PEGDA.

15. 10. The implant of claim 1, wherein the implant comprises about 0.225% (w / v) lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP).

16. The implant of claim 1 , wherein the implant is a biomimetic for peripheral nerve injury.

17. The implant of claim 1 , wherein the implant is a biomimetic for spinal cord injury.

18. The implant of claim 1 , wherein the implant is at least 5 mm in length.

19. a. determining the structure of the site of nerve injury or spinal cord injury; b. preparing a monomer solution of a photopolymerizable polymer; c. Filling the spacer with the monomer solution to control the length of the implant in the z-direction; d. printing a shell of the implant using microscale serial projection 3D printing, the shell being a layerless biomimetic nerve or spinal implant having a first end and a second end, the shell being biomimetic in shape with respect to host axonal tissue, and conforming to all or a portion of the structure of the nerve injury site or the spinal cord injury site; e. printing a layerless core of the implant using microscale serial projection 3D printing, wherein the core is a biomimetic nerve or spinal implant and comprises a plurality of linear channels that conform to all or a portion of the structure of the nerve injury site or the spinal cord injury site and mimic the host axonal tissue, the plurality of linear channels comprising a channel wall thickness of up to 100 micrometers, and the plurality of linear channels retain their pre-implant structure for at least four weeks after implantation; f. irradiating the implant with focused light to polymerize the photopolymerizable monomer solution into a polymer; Including, The implant body herein comprises polyethylene glycol diacrylate (PEGDA) or gelatin methacrylate (GelMa), A method for fabricating a biomimetic neural or spinal implant.

20. 20. The method of claim 19, wherein the step of irradiating with focused light is performed using a plurality of micromirrors.

21. The method of claim 19 , wherein the step of printing the shell comprises printing a plurality of linear microchannels.

22. 20. The method of claim 19, wherein the printing is performed in two stages.

23. 20. The method of claim 19, wherein the printing is done in two stages, each about 0.8 seconds long.

24. 20. The method of claim 19, wherein the concentrated light comprises UV light.

25. 20. The method of claim 19, wherein the printing is performed without scanning in both the X and Y directions.

26. 20. The method of claim 19, further comprising rinsing the implant with an antibiotic solution.

27. 20. The method of claim 19, further comprising fabricating the template from polystyrene fibers.

28. 28. The method of claim 27, wherein the polystyrene fibers are about 200 micrometers in diameter.

29. 28. The method of claim 27, wherein the polystyrene fibers are arranged in a hexagonal close-packed array.

30. 28. The method of claim 27, further comprising co-extruding the polystyrene fibers.

31. 28. The method of claim 27, further comprising bonding polystyrene end caps to the polystyrene fibers.

32. 20. The method of claim 19, further comprising the step of immersing the implant in propylene carbonate.

33. 20. The method of claim 19, further comprising rinsing the implant with ethanol.

34. The implant according to claim 1 , for implantation at the site of nerve damage or the site of spinal cord injury.

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